Dendrimer conjugates

ABSTRACT

The present invention relates to novel therapeutic and diagnostic dendrimers. In particular, the present invention is directed to dendrimer-linker conjugates, methods of synthesizing the same, compositions comprising the conjugates, as well as systems and methods utilizing the conjugates (e.g., in diagnostic and/or therapeutic settings (e.g., for the delivery of therapeutics, imaging, and/or targeting agents (e.g., in disease (e.g., cancer) diagnosis and/or therapy, pain therapy, etc.)). Accordingly, dendrimer-linker conjugates of the present invention may further comprise one or more components for targeting, imaging, sensing, and/or providing a therapeutic or diagnostic material and/or monitoring response to therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of pending U.S. patentapplication Ser. No. 12/403,179, filed Mar. 12, 2009, which claimspriority to expired U.S. Provisional Patent Application No. 61/035,949,filed Mar. 12, 2008, the contents of which are hereby incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under contract No.5RO1CA119409 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel therapeutic and diagnosticdendrimers. In particular, the present invention is directed todendrimer-linker conjugates, methods of synthesizing the same,compositions comprising the conjugates, as well as systems and methodsutilizing the conjugates (e.g., in diagnostic and/or therapeuticsettings (e.g., for the delivery of therapeutics, imaging, and/ortargeting agents (e.g., in disease (e.g., cancer) diagnosis and/ortherapy, pain therapy, etc.)). Accordingly, dendrimer-linker conjugatesof the present invention may further comprise one or more components fortargeting, imaging, sensing, and/or providing a therapeutic ordiagnostic material and/or monitoring response to therapy.

BACKGROUND OF THE INVENTION

Cancer remains the number two cause of mortality in the United States,resulting in over 500,000 deaths per year. Despite advances in detectionand treatment, cancer mortality remains high. New compositions andmethods for the imaging and treatment (e.g., therapeutic) of cancer mayhelp to reduce the rate of mortality associated with cancer.

Severe, chronic pain is observed a variety of subjects. For example,there exist large numbers of individuals with sever pain associated witharthritis, autoimmune disease, injury, cancer, and a host of otherconditions.

A vast number of different types of pain medications exist. For example,a number natural and synthetic alkaloids of opium (i.e., opioids) areuseful as analgesics for the treatment of severe pain. However, a numberof severe side effects associated with opioid and other pain medicationusage exist. For example, administration of opioid agonists oftenresults in intestinal dysfunction due to action of the opioid agonistupon the large number of receptors in the intestinal wall. Opioids aregenerally known to cause nausea and vomiting as well as inhibition ofnormal propulsive gastrointestinal function in animals, resulting inside effects such as constipation.

Pain medication (e.g., opioid)-induced side effects are a seriousproblem for patients being administered pain medications (e.g., opioidanalgesics) for both short term and long term pain management. Forinstance, more than 250,000 terminal cancer patients each year takeopioids, such as morphine, for pain relief, and about half of thosepatients experience severe constipation. At present, patients receivingopioid pain medications face the difficult choice of sufferingburdensome adverse effects (e.g., constipation) or ineffectiveanalgesia.

There exists a need for compositions, methods and systems for deliveringagents (e.g., diagnostic and/or therapeutic (e.g., cancer and/or paintherapeutics) to subjects that provide effective therapy (e.g., diseasetreatment, symptom relief, etc.) with reduced or eliminated sideeffects, even when administered in high doses.

SUMMARY

The present invention relates to novel therapeutic and diagnosticdendrimers. In particular, the present invention is directed todendrimer-linker conjugates, methods of synthesizing the same,compositions comprising the conjugates, as well as systems and methodsutilizing the conjugates (e.g., in diagnostic and/or therapeuticsettings (e.g., for the delivery of therapeutics, imaging, and/ortargeting agents (e.g., in disease (e.g., cancer) diagnosis and/ortherapy, pain therapy, etc.)). Accordingly, dendrimer-linker conjugatesof the present invention may further comprise one or more components fortargeting, imaging, sensing, and/or providing a therapeutic ordiagnostic material and/or monitoring response to therapy.

In particular, experiments conducted during the course of development ofembodiments for the present invention demonstrated formation and use oftherapeutic agents conjugated with dendrimers via a linker agent.Examples of linker agents include, but are not limited to, eliminationlinkers (e.g., 1,4 elimination linkers, 1,6 elimination linkers),cyclization based linkers, esterase sensitive linkers, glucoronidasesensitive linkers, hypoxia induced linkers (e.g., indolequinone), etc.

Accordingly, in certain embodiments, the present invention providesdendrimer conjugates comprising a linker, wherein said linker isconjugated to, for example, a G5 PAMAM dendrimer, a targeting agent, anda therapeutic compound. In some embodiments, the G5 PAMAM dendrimer isconjugated to an imaging agent.

The dendrimer conjugates are not limited to a particular type of linker.Examples of linker agents include, but are not limited to, eliminationlinkers (e.g., 1,4 elimination linkers, 1,6 elimination linkers),cyclization based linkers, esterase sensitive linkers, glucoronidasesensitive linkers, hypoxia induced linkers (e.g., indolequinone), etc.In some embodiments, the linker comprises a heteroaromatic nitrogencontaining compound. In some embodiments, the linker is a branchedself-elimination linker.

The dendrimer conjugates are not limited to particular therapeuticagents. In some embodiments, the therapeutic agent is Naloxone or aNaloxone pro-drug, or equivalent.

In certain embodiments, the present invention provides dendrimerconjugates comprising a G5 PAMAM dendrimer, a trigger, a linker, and atherapeutic compound. In some embodiments, the trigger is conjugated tothe dendrimer and to the linker. In some embodiments, the dendrimer isconjugated to the linker that is conjugated to the trigger and to thetherapeutic compound. In some embodiments, the enzyme labile bond linksthe trigger and the linker. In some embodiments, wherein upon cleavageof the labile bond the linkers self-degrade. In some embodiments,wherein cleavage of the linker is induced by hypoxia. In someembodiments, the linker is indolequinone.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 2 shows diagrams of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 3 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 4 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 5 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 6 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 7 shows the release of a therapeutic compound from a dendrimerconjugate in one embodiment of the invention.

FIG. 8 shows the release of a therapeutic compound from a dendrimerconjugate in one embodiment of the invention.

FIGS. 9A-9D show diagrams of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 10 shows the release of a therapeutic compound from esterasesensitive linker-dendrimer conjugate in one embodiment of the invention.

FIG. 11 shows examples of several (A, B, and C) elimination linkersdesigned for esterase triggered cleavage.

FIG. 12 shows the characterization of therapeutic compound release fromdendrimer conjugates of the present invention.

FIG. 13 shows dendrimer conjugate and methods of synthesizing the samein some embodiments of the invention.

FIG. 14 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 15 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 16 shows a diagram of a dendrimer conjugate provided in someembodiments of the present invention.

FIG. 17 shows an example of a dendrimer conjugate designed forglucuronidase triggered cleavage in one embodiment of the presentinvention.

FIG. 18 shows an example of a dendrimer conjugate designed for hypoxiainduced activation in one embodiment of the present invention.

FIG. 19 shows that, in some embodiments, a heteroaromatic nitro compoundpresent in a dendrimer conjugate of the present invention is reduced toeither an amine or a hydroxylamine, thereby triggering the spontaneousrelease of a therapeutic agent/drug.

FIG. 20 depicts the activation of a dendrimer conjugate comprisingeither a 1,4 or a 1,6 elimination linker in embodiments of the presentinvention.

FIG. 21 shows that a spacer (R2) can be used to decrease sterichindrance in a dendrimer conjugate in some embodiments of the presentinvention.

FIG. 22 depicts a dendrimer conjugate comprising a cyclization basedlinker in some embodiments of the present invention.

FIG. 23 depicts cyclization based linkers in some embodiments of theinvention.

FIG. 24 depicts a linker utilized in a dendrimer conjugate in someembodiments of the present invention.

FIG. 25 shows branched self-elimination linkers utilized in a dendrimerconjugate in some embodiments of the present invention.

FIGS. 26A and B depicts dendrimer conjugates provided in someembodiments of the present invention.

FIG. 27 shows a dendrimer comprising a simple ester (top portion offigure) and a dendrimer conjugate comprising an elimination linker(e.g., a 1, 6, elimination linker/spacer as shown in the bottomportion).

FIG. 28 shows a dendrimer conjugate comprising hydroxycamptothecin insome embodiments of the invention.

FIG. 29 shows a synthesis scheme for generating a dendrimer comprising ahypoxia induced linker.

FIG. 30 shows a synthesis scheme for generating a dendrimer comprising ahypoxia induced linker.

FIG. 31 shows a diagram depicting a mechanism of release of atherapeutic agent from a dendrimer comprising a hypoxia sensitivelinker.

FIG. 32 shows release of Naloxone from [dendrimer-indolequinonelinker-Naloxone prodrug] using the reductive enzyme DT-diaphorase.

FIG. 33 shows release of Naloxone from [dendrimer-indolequinonelinker-Naloxone prodrug] in human plasma under hypoxic conditions, butnot under normoxic conditions.

FIG. 34 shows hypoxia-induced release kinetics for Naloxone from[dendrimer-indolequinone linker-Naloxone prodrug] met or exceeded 6 mgNaloxone/hour at pO2 of 18 mmHg within fresh frozen plasma.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the terms “epithelial tissue” or “epithelium” refer tothe cellular covering of internal and external surfaces of the body,including the lining of vessels and other small cavities. It consists ofcells joined by small amounts of cementing substances. Epithelium isclassified into types on the basis of the number of layers deep and theshape of the superficial cells.

As used herein, the term “normal epithelium of prostate” refers toprostate epithelium that does not show any detectable indication ofcancerous or pre-cancerous conditions.

As used herein, the term “cancerous epithelium of prostate” refers toprostate epithelium that shows a detectable indication of cancerous orpre-cancerous conditions.

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancermay also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has received apreliminary diagnosis (e.g., a CT scan showing a mass) but for whom aconfirmatory test (e.g., biopsy and/or histology) has not been done orfor whom the stage of cancer is not known. The term further includespeople who once had cancer (e.g., an individual in remission). A“subject suspected of having cancer” is sometimes diagnosed with cancerand is sometimes found to not have cancer.

As used herein, the terms “prostate specific membrane antigent” or“PSMA” refer to a membrane-bound epitope, originally identified byHoroszewicz et al. (See, e.g., Horoszewicz et al., Anticancer Res 7,927, (1987); van Steenbrugge et al., Urol Res 17, 71 (1989); Carter etal., Proc Natl Acad Sci USA. 93(2): 749 (1996)), selectively expressedin epithelial cells of prostatic origin. Small amounts of PSMAexpression have been detected in a variety of tumors (See, e.g., Changet al., Clin Cancer Res 5, 2674 (1999)).

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention. A “preliminary diagnosis” is one based only on visual(e.g., CT scan or the presence of a lump) and antigen tests (e.g.,PSMA).

As used herein, the term “initial diagnosis” refers to a test result ofinitial cancer diagnosis that reveals the presence or absence ofcancerous cells (e.g., using a biopsy and histology).

As used herein, the term “prostate tumor tissue” refers to canceroustissue of the prostate.

In some embodiments, the prostate tumor tissue is “post surgicalprostate tumor tissue.”

As used herein, the term “post surgical tumor tissue” refers tocancerous tissue (e.g., prostate tissue) that has been removed from asubject (e.g., during surgery).

As used herein, the term “identifying the risk of said tumormetastasizing” refers to the relative risk (e.g., the percent chance ora relative score) of a tumor (e.g., prostate tumor tissue)metastasizing.

As used herein, the term “identifying the risk of said tumor recurring”refers to the relative risk (e.g., the percent chance or a relativescore) of a tumor (e.g., prostate tumor tissue) recurring in the sameorgan as the original tumor (e.g., prostate).

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental expose, and previous incidents of cancer,preexisting non-cancer diseases, and lifestyle.

As used herein, the term “characterizing cancer in subject” refers tothe identification of one or more properties of a cancer sample in asubject, including but not limited to, the presence of benign,pre-cancerous or cancerous tissue and the stage of the cancer. Cancersmay be characterized by identifying cancer cells with the compositionsand methods of the present invention. For example, cancers may becharacterized by detecting expression of PSMA with the compositions andmethods of the present invention.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor, whether the tumor has spread to otherparts of the body and where the cancer has spread (e.g., within the sameorgan or region of the body or to another organ).

Several staging methods are commonly used for cancer (e.g, prostatecancer). A common classification of the spread of prostate cancer wasdeveloped by the American Urological Association (AUA). The AUA systemdivides prostate tumors into four stages, A to D. Stage A, microscopiccancer within prostate, is further subdivided into stages A1 and A2.Sub-stage A1 is a well-differentiated cancer confined to one site withinthe prostate. Treatment is generally observation, radical prostatectomy,or radiation. Sub-stage A2 is a moderately to poorly differentiatedcancer at multiple sites within the prostate. Treatment is radicalprostatectomy or radiation. Stage B, palpable lump within the prostate,is also further subdivided into sub-stages B1 and B2. In sub-stage B1,the cancer forms a small nodule in one lobe of the prostate. Insub-stage B2, the cancer forms large or multiple nodules, or occurs inboth lobes of the prostate. Treatment for sub-stages B1 and B2 is eitherradical prostatectomy or radiation. Stage C is a large cancer massinvolving most or all of the prostate and is also further subdividedinto two sub-stages. In sub-stage C1, the cancer forms a continuous massthat may have extended beyond the prostate. In sub-stage C2, the cancerforms a continuous mass that invades the surrounding tissue. Treatmentfor both these sub-stages is radiation with or without drugs to addressthe cancer. The fourth stage, Stage D is metastatic cancer and is alsosubdivided into two sub-stages. In sub-stage D1, the cancer appears inthe lymph nodes of the pelvis. In sub-stage D2, the cancer involvestissues beyond lymph nodes. Treatment for both of these sub-stages issystemic drugs to address the cancer as well as pain.

As used herein, the term “GLEASON score” refers to a histologic gradethat refers to the microscopic characteristics of malignant prostatictumor. Individual areas receive a grade from 1 to 5. Cells that are welldifferentiated are given a low grade; poorly differentiated cells aregiven a high grade. A primary grade is assigned to the pattern occupyingthe greatest area of the specimen and a secondary grade is assigned tothe second-largest affected area. These two grades are then addedtogether for an overall Gleason score (or sum). The mostwell-differentiated cancer would receive a Gleason score of 2 (1+1),while the most poorly differentiated cancer would receive a Gleasonscore of 10 (5+5).

Staging of prostate cancer can also be based on the revised criteria ofTNM staging by the American Joint Committee for Cancer (AJCC) publishedin 1988. Staging is the process of describing the extent to which cancerhas spread from the site of its origin. It is used to assess a patient'sprognosis and to determine the choice of therapy. The stage of a canceris determined by the size and location in the body of the primary tumor,and whether it has spread to other areas of the body. Staging involvesusing the letters T, N and M to assess tumors by the size of the primarytumor (T); the degree to which regional lymph nodes (N) are involved;and the absence or presence of distant metastases (M)—cancer that hasspread from the original (primary) tumor to distant organs or distantlymph nodes. Each of these categories is further classified with anumber 1 through 4 to give the total stage. Once the T, N and M aredetermined, a “stage” of I, II, III or IV is assigned. Stage I cancersare small, localized and usually curable. Stage H and III cancerstypically are locally advanced and/or have spread to local lymph nodes.Stage 1V cancers usually are metastatic (have spread to distant parts ofthe body) and generally are considered inoperable.

As used herein, the term “characterizing tissue in a subject” refers tothe identification of one or more properties of a tissue sample (e.g.,including but not limited to, the presence of cancerous tissue, thepresence of pre-cancerous tissue that is likely to become cancerous, andthe presence of cancerous tissue that is likely to metastasize). In someembodiments, tissues are characterized detecting expression of PSMA withthe compositions and methods of the present invention.

As used herein, the term “reagent(s) capable of specifically detectingPSMA expression” refers to reagents used to detect the expression andlocation of PSMA. Examples of suitable reagents include but are notlimited to, the dendrimers of the present invention.

As used herein, the term “instructions for using said kit for detectingcancer in said subject” includes instructions for using the reagentscontained in the kit for the detection and characterization of cancer ina sample from a subject.

As used herein, the terms “computer memory” and “computer memory device”refer to any storage media readable by a computer processor. Examples ofcomputer memory include, but are not limited to, RAM, ROM, computerchips, digital video disc (DVDs), compact discs (CDs), hard disk drives(HDD), and magnetic tape.

As used herein, the term “computer readable medium” refers to any deviceor system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tape and servers for streaming media over networks.

As used herein, the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of cancer (e.g., asdetermined by the diagnostic methods of the present invention) on asubject's future health (e.g., expected morbidity or mortality, thelikelihood of getting cancer, and the risk of metastasis).

As used herein, the term “non-human animals” refers to all non-humananimals including, but not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

As used herein, the term “gene transfer system” refers to any means ofdelivering a composition comprising a nucleic acid sequence to a cell ortissue. For example, gene transfer systems include, but are not limitedto, vectors (e.g., retroviral, adenoviral, adeno-associated viral, andother nucleic acid-based delivery systems), microinjection of nakednucleic acid, dendrimers, polymer-based delivery systems (e.g.,liposome-based and metallic particle-based systems), biolisticinjection, and the like. As used herein, the term “viral gene transfersystem” refers to gene transfer systems comprising viral elements (e.g.,intact viruses, modified viruses and viral components such as nucleicacids or proteins) to facilitate delivery of the sample to a desiredcell or tissue. As used herein, the term “adenovirus gene transfersystem” refers to gene transfer systems comprising intact or alteredviruses belonging to the family Adenoviridae.

As used herein, the term “site-specific recombination target sequences”refers to nucleic acid sequences that provide recognition sequences forrecombination factors and the location where recombination takes place.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-aminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “transgene” refers to a heterologous gene thatis integrated into the genome of an organism (e.g., a non-human animal)and that is transmitted to progeny of the organism during sexualreproduction.

As used herein, the term “transgenic organism” refers to an organism(e.g., a non-human animal) that has a transgene integrated into itsgenome and that transmits the transgene to its progeny during sexualreproduction.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encodedin a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of mRNA. Gene expression can beregulated at many stages in the process. “Up-regulation” or “activation”refers to regulation that increases the production of gene expressionproducts (i.e., RNA or protein), while “down-regulation” or “repression”refers to regulation that decrease production. Molecules (e.g.,transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics (includingaltered nucleic acid sequences) when compared to the wild-type gene orgene product.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in a cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotideor polynucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 15 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“5′-A-G-T-3′,” is complementary to the sequence “5′-T-C-A-3′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is a nucleic acid molecule that at leastpartially inhibits a completely complementary nucleic acid molecule fromhybridizing to a target nucleic acid is “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous nucleic acid molecule to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target that issubstantially non-complementary (e.g., less than about 30% identity); inthe absence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985)). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 (1972)).Other nucleic acids will not be replicated by this amplification enzyme.Similarly, in the case of T7 RNA polymerase, this amplification enzymehas a stringent specificity for its own promoters (Chamberlin et al.,Nature 228:227 (1970)). In the case of T4 DNA ligase, the enzyme willnot ligate the two oligonucleotides or polynucleotides, where there is amismatch between the oligonucleotide or polynucleotide substrate and thetemplate at the ligation junction (Wu and Wallace, Genomics 4:560(1989)). Finally, Taq and Pfu polymerases, by virtue of their ability tofunction at high temperature, are found to display high specificity forthe sequences bounded and thus defined by the primers; the hightemperature results in thermodynamic conditions that favor primerhybridization with the target sequences and not hybridization withnon-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press(1989)).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target.”In contrast, “background template” is used in reference to nucleic acidother than sample template that may or may not be present in a sample.Background template is most often inadvertent. It may be the result ofcarryover, or it may be due to the presence of nucleic acid contaminantssought to be purified away from the sample. For example, nucleic acidsfrom organisms other than those to be detected may be present asbackground in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” refers to the region of nucleic acidbounded by the primers. Thus, the “target” is sought to be sorted outfrom other nucleic acid sequences. A “segment” is defined as a region ofnucleic acid within the target sequence.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The terms “in operable combination,” “in operable order,” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

“Amino acid sequence” and terms such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

The term “native protein” as used herein to indicate that a protein doesnot contain amino acid residues encoded by vector sequences; that is,the native protein contains only those amino acids found in the proteinas it occurs in nature. A native protein may be produced by recombinantmeans or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58 (1989)).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52(1989)).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Vectorsare often derived from plasmids, bacteriophages, or plant or animalviruses.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher (or greater) than thatobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA are measured using any of a number of techniques known to thoseskilled in the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the mRNA-specificsignal observed on Northern blots). The amount of mRNA present in theband corresponding in size to the correctly spliced transgene RNA isquantified; other minor species of RNA which hybridize to the transgeneprobe are not considered in the quantification of the expression of thetransgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 (1973)),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

As used herein, the term “selectable marker” refers to the use of a genethat encodes an enzymatic activity that confers the ability to grow inmedium lacking what would otherwise be an essential nutrient (e.g. theHIS3 gene in yeast cells); in addition, a selectable marker may conferresistance to an antibiotic or drug upon the cell in which theselectable marker is expressed. Selectable markers may be “dominant”; adominant selectable marker encodes an enzymatic activity that can bedetected in any eukaryotic cell line. Examples of dominant selectablemarkers include the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) that confers resistance to the drugG418 in mammalian cells, the bacterial hygromycin G phosphotransferase(hyg) gene that confers resistance to the antibiotic hygromycin and thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) that confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that their use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene that is used in conjunction withtk⁻ cell lines, the CAD gene that is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene that is used in conjunction withhprt⁻ cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NewYork (1989) pp. 16.9-16.15.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, transformed celllines, finite cell lines (e.g., non-transformed cells), and any othercell population maintained in vitro.

As used herein, the term “eukaryote” refers to organisms distinguishablefrom “prokaryotes.” It is intended that the term encompass all organismswith cells that exhibit the usual characteristics of eukaryotes, such asthe presence of a true nucleus bounded by a nuclear membrane, withinwhich lie the chromosomes, the presence of membrane-bound organelles,and other characteristics commonly observed in eukaryotic organisms.Thus, the term includes, but is not limited to such organisms as fungi,protozoa, and animals (e.g., humans).

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water,crystals and industrial samples. Such examples are not however to beconstrued as limiting the sample types applicable to the presentinvention.

As used herein, the term “NAALADase inhibitor” refers to any one of amultitude of inhibitors for the neuropeptidase NAALADase(N-acetylated-alpha linked acidic dipeptidase). Such inhibitors ofNAALADase have been well characterized. For example, an inhibitor can beselected from the group comprising, but not limited to, those found inU.S. Pat. No. 6,011,021, herein incorporated by reference in itsentirety.

As used herein, the term “nanodevice” or “nanodevices” refer, generally,to compositions comprising dendrimers of the present invention. As such,a nanodevice may refer to a composition comprising a dendrimer and metalnanoparticles (e.g., iron oxide nanoparticles (e.g., poly(styrenesulfonate) (PSS)-coated iron oxide nanoparticles)) of the presentinvention that may contain one or more functional groups (e.g., atherapeutic agent) conjugated to the dendrimer. A nanodevice may alsorefer to a composition comprising two or more different dendrimers ofthe present invention.

As used herein, the term “degradable linkage,” when used in reference toa polymer (e.g., PEG-hRNase conjugate of the present invention), refersto a conjugate that comprises a physiologically cleavable linkage (e.g.,a linkage that can be hydrolyzed (e.g., in vivo) or otherwise reversed(e.g., via enzymatic cleavage). Such physiologically cleavable linkagesinclude, but are not limited to, ester, carbonate ester, carbamate,sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal linkages (See,e.g., U.S. Pat. No. 6,838,076, herein incorporated by reference in itsentirety). Similarly, the conjugate may comprise a cleavable linkagepresent in the linkage between the polymer and hRNase, or, may comprisea cleavable linkage present in the polymer itself (e.g., such that whencleaved, a small portion of the polymer remains on the hRNase molecule)(See, e.g., U.S. Pat. App. Nos. 20050158273 and 20050181449, each ofwhich is herein incorporated by reference in its entirety). For example,a PEG polymer comprising an ester linkage can be utilized forconjugation to hRNase to create a PEG-hRNase conjugate (See, e.g.,Kuzlowski et al., Biodrugs, 15, 419-429 (2001). A conjugate thatcomprises a degradable linkage of the present invention is capable ofgenerating hRNase that is free (e.g., completely or partially free) ofthe polymer (e.g., in vivo after hydrolysis of the linkage).

A “physiologically cleavable” or “hydrolysable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include but are not limited tocarboxylate ester, phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond(e.g., typically a covalent bond) that is substantially stable in water(i.e., does not undergo hydrolysis under physiological conditions to anyappreciable extent over an extended period of time). Examples ofhydrolytically stable linkages include, but are not limited to,carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides,urethanes, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel therapeutic and diagnosticdendrimers. In particular, the present invention is directed todendrimer-linker conjugates, methods of synthesizing the same,compositions comprising the conjugates, as well as systems and methodsutilizing the conjugates (e.g., in diagnostic and/or therapeuticsettings (e.g., for the delivery of therapeutics, imaging, and/ortargeting agents (e.g., in disease (e.g., cancer) diagnosis and/ortherapy, pain therapy, etc.)). Accordingly, dendrimer-linker conjugatesof the present invention may further comprise one or more components fortargeting, imaging, sensing, and/or providing a therapeutic ordiagnostic material and/or monitoring response to therapy.

Accordingly, in some embodiments, the present invention provides alinker conjugated to a targeting agent (e.g., that can be conjugated toa dendrimer (e.g., for specific targeting of the dendrimer)). Thus, insome embodiments, the present invention provides methods of synthesizingdendrimer conjugates (e.g., PAMAM dendrimers) comprising linkers (e.g.,conjugated to a trigger moiety, therapeutic moiety and/or other type ofmoiety) compositions comprising the same, and methods of using the samein the diagnosis, imaging and treatment of disease (e.g., cancer,inflammatory disease, chronic pain, etc.).

The present invention provides a multiplicity of linkers and targetingagents that find use in the present invention. Indeed, the presentinvention is not limited to any particular linker or to any particulartargeting agent or to any particular dendrimer. In some embodiments, thepresent invention provides a dendrimer conjugated to a linker that isconjugated to a targeting agent, and methods of generating and using thesame (e.g., to treat cancer, pain and/or inflammation, etc.). In someembodiments, a dendrimer conjugated to a linker that is conjugated to atargeting agent decreases the number of conjugation steps required toform a dendrimer (e.g., a dendrimer conjugate (e.g., a dendrimerconjugated to a targeting agent, imaging agent, therapeutic agent and/orsensing agent)). For example, in some embodiments, the present inventionprovides a customizable dendrimer wherein one or a plurality of linkers(e.g. attached to one or a plurality of targeting agents, triggeringagents and/or therapeutic agents) are conjugated to a dendrimer, therebydecreasing the number of conjugation steps used to form a dendrimer(e.g., versus a dendrimer that is conjugated to a targeting moiety inone step and that is separately conjugatd to a linker (e.g., comprisinga therapeutic agent, imaging agent, sensing agent or other moiety) in anadditional conjugation step). In some embodiments, a linker conjugatedto one or more targeting agents is conjugated to one or more additionalmoieties including, but not limited to, a therapeutic agent, atriggering agent, an imaging agent, a sensing agent, etc. Thus, in someembodiments, the present invention provides a dendrimer with increasedload capacity (e.g., increased load of therapeutic, imaging agent, etc.on the dendrimer). In some embodiments, two or more linkers (e.g.,conjugated to one or a plurality of targeting agents) are conjugated toa dendrimer via the same or different linkage (e.g., covalent linkage).

Several different schemes were evaluated for generating dendrimerconjugates wherein a dendrimer is conjugated to one or more linkers thatcomprise multiple sites for binding (e.g., covalent binding) moieties.For example, in one embodiment, a linker may comprise a chemicalstructure that allows conjugation of a targeting moiety and atherapeutic compound to the linker. Thus, in some embodiments, adendrimer conjugate of the present invention permits control of thestoichiometry between targeting agent and therapeutic compound (e.g.,generation of one to one ratio, two to one ratio, one to two ratio, oneto three ratio etc. between targeting and therapeutic moieties).

In some embodiments, a dendrimer conjugated to a linker that isconjugated to a targeting agent and/or therapeutic comprises a linkerthat is configured to be irreversibly degraded (e.g., that isnon-reversible (e.g., that permits drug delivery at the correct timeand/or at the correct place)).

In some embodiments, the present invention provides a dendrimerconjugate as shown in FIG. 1. For example, FIG. 1 shows a targetingagent (T.A.) conjugated to a linker that is also conjugated to a drug,wherein the linker conjugated to a drug and targeting agent isconjugated to a dendrimer conjugated to an imaging agent. In someembodiments, the present invention provides a dendrimer conjugate asshown in FIG. 2 (e.g., possessing targeted anticancer therapeuticmoiety). For example, FIG. 2 shows several structures of dendrimerconjugates, wherein R1, R2, R3 and R4 are each independently selectedfrom hydrogen, halogen, and alkyl. In some embodiments, the alkyl isstraight or cyclic, unsubstituted or substituted (e.g., by from 1 to 4substituents (e.g., selected from the group comprising, but not limitedto, halogen, amino, monoalkylamino, dialkylamino, hydroxy, alkoxy,nitro, aryl, cyano, carboxyl, carboxamide, monoalkylcarboxamide,dialkylcarboxamide, thiol, thioalkyl and sulfonic acid. In someembodiments, the “U” moiety is present or absent. In some embodiments,when the “U” moiety is absent, one of the R1, R2, R3 and/or R4 groups islinked to a targeting agent through a linker and/or spacer. In someembodiments, R5 is an alkyl (e.g., that is straight chained, branched,cyclic (e.g., that is substituted or unsubtituted)). In someembodiments, R6 is a hydrogen or an alkyl (e.g., of 1-4 carbons (e.g.,that are straight chained or cyclic (e.g., that is substituted orunsubstituted)). In some embodiments, Ra, Rb, Rc, Rd and Re are eachindependently selected from hydrogen, halogen, and alkyl. In someembodiments, the alkyl is straight or cyclic, unsubstituted orsubstituted (e.g., by from 1 to 4 substituents (e.g., selected fromhalogen, amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, nitro,aryl, cyano, carboxyl, carboxamide, monoalkylcarboxamide,dialkylcarboxamide, thiol, thioalkyl and sulfonic acid. In someembodiments, the “U” moiety is present or absent. In some embodiments,when the “U” moiety is absent, one of the Ra, Rb, Rc, Rd and Re groupsis linked to a targeting agent through a linker and/or spacer. In someembodiments, “Y” is an oxygen atom. In some embodiments, “Y” is twohydrogen atoms. In some embodiments, G5 is a generation five poly(amidoamine) (PAMAM) dendrimer (e.g., conjugated to one or more imagingagents (e.g., FITC, etc.), although higher (e.g., G6, G7, G8, G9, G10 orhigher, or lower, G4, G3, or G2 dendrimers may also be used. In someembodiments, “W” is a linker comprising 1-8 carbon and/or nitrogen atoms(e.g., straight chanined, branched, or cyclic, unsubstituted orsubstituted by “R” groups as described above.

In some embodiments, the present invention provides a dendrimerconjugate as shown in FIGS. 3 and 4. In particular, a dendrimerconjugate as shown in FIG. 3 comprises a dendrimer (e.g., a G5 PAMAMdendrimer conjugated to an imaging agent (e.g., FITC) and/or targetingagent) conjugated to a trigger molecule that is conjugated to a linkerthat is conjugated to a therapeutic. A dendrimer conjugate as shown inFIG. 4 comprises a dendrimer (e.g., a G5 PAMAM dendrimer conjugated toan imaging agent (e.g., FITC) and/or targeting agent) conjugated to alinker that is conjugated to a trigger and to a therapeutic moiety. Theconjugates of FIGS. 3 and 4 are configured to be non-toxic to normalcells. For example, the conjugates are configured in such a way so as torelease their therapeutic agent only at a specific, targeted site (e.g.,through activation of a trigger molecule that in to leads to release ofthe therapeutic agent) For example, once a conjugate arrives at a targetsite in a subject (e.g., a tumor, or a site of inflammation), componentsin the target site (e.g., a tumor associated factor, or an inflammatoryor pain associated factor) interacts with the trigger moiety therebyinitiating cleavage of this unit from the linker. In some embodiments,once the trigger is cleaved from the linker (e.g., by a targetassociated moiety, the linker proceeds through spontaneous chemicalbreakdown thereby releasing the therapeutic agent at the target site(e.g., in its active form). The present invention is not limited to anyparticular target associated moiety (e.g., that interacts with andinitiates cleavage of a trigger). In some embodiments, the targetassociated moiety is a tumor associated factor (e.g., an enzyme (e.g.,glucuronidase and/or plasmin), a cathepsin, a matrix metalloproteinase,a hormone receptor (e.g., integrin receptor, hyaluronic acid receptor,luteinizing hormone-releasing hormone receptor, etc.), cancer and/ortumor specific DNA sequence), an inflammatory associated factor (e.g.,chemokine, cytokine, etc.) or other moiety.

Although an understanding of a mechanism of action is not necessary topractice the present invention, and the present invention is not limitedto any particular mechanism of action, in some embodiments, a dendrimerconjugate as described in FIG. 3 or 4 provides a therapeutic to a siteby a mechanism as shown in FIG. 5 or 6. For example, as shown in FIG. 5,a dendrimer conjugate comprising a dendrimer (e.g., a G5 PAMAM dendrimerconjugated to an imaging agent (e.g., FITC) and/or targeting agent)conjugated to a trigger molecule that is conjugated to a linker that isconjugated to a therapeutic (A) interacts with a target associatedmoiety thereby activating the trigger and initiating cleavage of same,releasing the linker therapeutic drug conjugate. Once cleavage of thetrigger occurs, the linker (B) proceeds through a spontaneous chemicalbreakdown at the target site, releasing (e.g., irreversibly releasing)the therapeutic drug at the target site. In some embodiments, as shownin FIG. 6, a dendrimer conjugate comprising a dendrimer (e.g., a G5PAMAM dendrimer conjugated to an imaging agent (e.g., FITC) and/ortargeting agent) conjugated to a linker that is conjugated to a triggerand to a therapeutic moiety (A) interacts with a target associatedmoiety thereby activating the trigger and initiating cleavage of same,releasing a dendrimer-linker-therapeutic moiety from the trigger. Oncecleavage of the trigger occurs, the linker (B) proceeds through aspontaneous chemical breakdown (e.g., to a point where the therapeuticdrug is released from the dendrimer linker conjugate) at the targetsite, releasing (e.g., irreversibly releasing) the therapeutic drug atthe target site. Several design processes for generating a dendrimerconjugate comprising a trigger are shown in FIGS. 7 and 8.

The dendrimer conjugates shown in FIGS. 3 and 4 are not limited to anyparticular dendrimer. Indeed, the conjugates may comprise a variety ofdifferent types of dendrimers. In some embodiments, the dendrimer is aPAMAM dendrimer (e.g., G3, G5 or G7 dendrimer). In some embodiments, oneor more amino groups present on the dendrimer are linked (e.g., througha covalent bond) to one or more targeting agents (e.g., folic acid)and/or imaging agents (e.g., FITC) (e.g., as described in U.S. Pat. Nos.6,471,968 and 7,078,461; U.S. Patent Pub. Nos. 20020165179 and20070041934 and WO 06/033766, each of which is hereby incorporated byreference in its entirety for all purposes).

In some embodiments, the present invention provides a dendrimerconjugate as shown in FIG. 9. In particular, a dendrimer conjugate asshown in FIG. 9 comprises a dendrimer (e.g., a G5 PAMAM dendrimerconjugated to an imaging agent (e.g., FITC) and/or targeting agent)conjugated to a trigger molecule that is conjugated to a linker that isconjugated to a therapeutic, or a dendrimer (e.g., a G5 PAMAM dendrimerconjugated to an imaging agent (e.g., FITC) and/or targeting agent)conjugated to a linker that is conjugated to a trigger and to atherapeutic moiety.). For example, FIG. 9 shows several structures ofdendrimer conjugates, wherein R1, R2, R3 and R4 are each independentlyselected from hydrogen, halogen, and alkyl. In some embodiments, thealkyl is straight or cyclic, unsubstituted or substituted (e.g., by from1 to 4 substituents (e.g., selected from the group comprising, but notlimited to, halogen, amino, monoalkylamino, dialkylamino, hydroxy,alkoxy, nitro, aryl, cyano, carboxyl, carboxamide, monoalkylcarboxamide,dialkylcarboxamide, thiol, thioalkyl and sulfonic acid. In someembodiments, R5 is an alkyl that is straight, branched or cyclic, thatis unsubstituted or substituted. In some embodiments, R6 is a hydrogenor alkyl of 1-4 carbons that are straight, branched or cyclic, that isunsubstituted or substituted. In some embodiments, the two R6 areconnected together to form a ring of 306 members. In some embodiments,R′, R″, R′″ and R″″ are each independently selected from hydrogen,halogen, and alkyl. In some embodiments, the alkyl is straight orcyclic, unsubstituted or substituted (e.g., by from 1 to 4 substituents(e.g., selected from the group comprising, but not limited to, halogen,amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, nitro, aryl,cyano, carboxyl, carboxamide, monoalkylcarboxamide, dialkylcarboxamide,thiol, thioalkyl and sulfonic acid. In some embodiments, X, X2 and X3are either oxygen or “NR”, wherein “N” is a nitrogen atom, and “R” is analkyl that is straight or branched or cyclic (e.g., substituted orunsubstituted). In some embodiments, “Y” is an oxygen atom or twohydrogen atoms. In some embodiments, A-B is an ethylene group (e.g.,unsubstituted or substituted by alkyls (e.g., straight or cyclic). Insome embodiments, A-B are connected by a carbon chain (e.g., of 2, 3, 4,5, or more carbons) and/or hetero atoms (e.g., forming a saturated orunsaturated aromatic ring structure (e.g., comprising substituents suchas R1, R2, R3 and R4). In some embodiments, G5 is a dendrimer (e.g., aG5 PAMAM dendrimer conjugated to an imaging agent (e.g., FITC) and/ortargeting agent). As described herein, the present invention is notlimited to any particular dendrimer. In some embodiments, “W” is alinker (e.g., comprising a carbon or nitrogen chain (e.g., 2, 3, 4, 5,6, 7, 8, 9, or more carbons or nitrogens (e.g., straight or branched orcyclic (e.g., substituted or unsubstituted (e.g., with R groups asdescribed above))).

The present invention is not limited by the type of dendrimer conjugate(e.g., comprising a trigger) for use in treating a subject. For example,the present invention contemplates dendrimer conjugates comprising oneor more anticancer prodrugs developed for site specific conversion todrug based on tumor associated factors (e.g., hypoxia and pH,tumor-associated enzymes, and/or receptors). In some embodiments,dendrimer conjugates of the present invention are configured such that aprodrug (e.g., anticancer prodrug) is conjugated to a linker that isfurther conjugated to a targeting moiety (e.g., that targets theconjugate to a site of cancer and/or tumor). Although an understandingof the mechanism is not necessary for the present invention, and thepresent invention is not limited to any particular mechanism of action,in some embodiments, a trigger component serves as a precursor forsite-specific activation. For example, in some embodiments, once atumor-associated factor recognizes a trigger, cleavage and/or processingof the trigger is induced. The present invention is not limited to anyparticular trigger or to any particular cleavage and/or processing ofthe trigger. In some embodiments, the present invention provides adendrimer conjugate comprising a trigger that is sensitive to (e.g., iscleaved by) and/or that associates with a tumor associated enzyme. Insome embodiments, the present invention provides a dendrimer conjugatecomprising a trigger that is sensitive to (e.g., is cleaved by) and/orthat associates with a glucuronidase. Glucuronic acid can be attached toseveral anticancer drugs via various linkers. These anticancer drugsinclude, but are not limited to, doxorubicin, paclitaxel, docetaxel,5-fluorouracil, 9-aminocamtothecin, as well as other drugs underdevelopment. These prodrugs are generally stable at physiological pH andare significantly less toxic than the parent drugs. In some embodiments,dendrimer conjugates comprising anticancer prodrugs find use fortreating necrotic tumors (e.g., that liberate β-glucuronidase) or forADEPT with antibodies that can deliver β-glucuronidase to target tumorcells. An example of an anticancer prodrug is shown in Figure suchprodrugs is shown below:

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger that is sensitive to (e.g., is cleavedby) and/or that associates with a protease. The present invention is notlimited to any particular protease. In some embodiments, the protease isa cathepsin. In some embodiments, a trigger comprises a Lys-Phe-PABCmoiety (e.g., that acts as a trigger). In some embodiments, aLys-Phe-PABC moiety linked to doxorubicin, mitomycin C, and paclitaxelare utilized as a trigger-therapeutic conjugate in a dendrimer conjugateprovided herein (e.g., that serve as substrates for lysosomal cathepsinB or other proteases expressed (e.g., overexpressed) in tumor cells. Insome embodiments, utilization of a 1,6-elimination spacer/linker isutilized (e.g., to permit release of therapeutic drug post activation oftrigger).

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger that is sensitive to (e.g., is cleavedby) and/or that associates with plasmin. The serine protease plasmin isover expressed in many human tumor tissues. Tripeptide specifiers (e.g.,including, but not limited to, Val-Leu-Lys) have been identified andlinked to anticancer drugs through elimination or cyclization linkers.

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger that is sensitive to (e.g., is cleavedby) and/or that associates with a matrix metalloproteases (MMPs). Insome embodiments, the present invention provides a dendrimer conjugatecomprising a trigger that is sensitive to (e.g., is cleaved by) and/orthat associates with β-Lactamase (e.g., a β-Lactamase activatedcephalosporin-based prodrug).

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger that is sensitive to (e.g., is cleavedby) hypoxia (e.g., indolequinone) (e.g., as described in Example 8).Hypoxia is a feature of several disease states, including cancer,inflammation and rheumatoid arthritis. Advances in the chemistry ofbioreductive drug activation have led to the design of varioushypoxia-selective drug delivery systems in which the pharmacophores ofdrugs are masked by reductively cleaved groups. In some embodiments, adendrimer conjugate of the present invention utilizes a quinone, N-oxideand/or (hetero)aromatic nitro groups. For example, a quinone present ina dendrimer conjugate of the present invention is reduced to phenolunder hypoxia conditions, with spontaneous formation of lactone thatserves as a driving force for drug release (e.g., as shown in FIG. 18).In some embodiments, the quinone is an indolequinone. In someembodiments, a heteroaromatic nitro compound present in a dendrimerconjugate of the present invention is reduced to either an amine or ahydroxylamine, thereby triggering the spontaneous release of atherapeutic agent/drug (e.g., as shown in FIG. 19).

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger that is sensitive to (e.g., is cleavedby) and/or activated by a receptor (e.g., expressed on a target cell(e.g., a tumor cell)). Thus, in some embodiments, a dendrimer conjugatecomprises a receptor binding motif conjugated to a therapeutic agent(e.g., cytotoxic drug) thereby providing target specificity. Examplesinclude, but are not limited to, a dendrimer conjugate comprising aprodrug (e.g., of doxorubicin and/or paclitaxel) targeting integrinreceptor, a hyaluronic acid receptor, and/or a hormone receptor.

In some embodiments, the present invention provides a dendrimerconjugate comprising a trigger that is sensitive to (e.g., is cleavedby) and/or activated by a nucleic acid. Nucleic acid triggered catalyticdrug release can be utilized in the design of chemotherapeutic agents.Thus, in some embodiments, disease specific nucleic acid sequence isutilized as a drug releasing enzyme-like catalyst (e.g., via complexformation with a complimentary catalyst-bearing nucleic acid and/oranalog).

In some embodiments, the present invention provides a dendrimerconjugate comprising a linker that connects a trigger to a therapeuticcompound. In some embodiments, the linker is configured such that itsdecomposition leads to the liberation (e.g., non-reversible liberation)of the therapeutic agent (e.g., at the target site (e.g., site of tumor,or inflammatory site)). The linker may influence multiplecharacteristics of a dendrimer conjugate including, but not limited to,properties of the therapeutic agent (e.g., stability, pharmacokinetic,organ distribution, bioavailability, and/or enzyme recognition (e.g.,when the therapeutic agent (e.g., prodrug)) is enzymaticallyactivated)).

In some embodiments, the linker is an elimination linker. For example,in some embodiments, in a dendrimer conjugate of the present invention,when a trigger is cleaved (e.g., enzymatically and/or chemically), aphenol or an aniline promotes a facile 1,4 or 1,6 elimination, followedby release of a CO₂ molecule and the unmasked therapeutic agent (e.g.,drug) (See, e.g., FIG. 20). In some embodiments, a dendrimer conjugateof the present invention utilizes this configuration and/or strategy tomask one or more hydroxyl groups and/or amino groups of the therapeuticagents. In some embodiments, a linker present within a dendrimerconjugate of the present invention is fine tuned (e.g., to optimizestability and/or drug release from the conjugate). For example, thesizes of the aromatic substituents can be altered (e.g., increased ordecreased) and/or alkyl substitutions at the benzylic position may bemade to alter (e.g., increase or decrease) degradation of the linkerand/or release of the therapeutic agent (e.g., prodrug). In someembodiments, elongated analogs (e.g., double spacers) are used (e.g., todecrease steric hindrance (e.g., for large therapeutic agents (e.g., SeeFIG. 21))). In some embodiments, a dendrimer conjugate of the presentinvention comprises an enol based linker (e.g., that undergoes anelimination reaction to release therapeutic agent (e.g., prodrug)).

In some embodiments, the linker is a cyclization based linker. Forexample, one configuration for this approach is shown in FIG. 22. Anucleophilic group (e.g., OH or NHR) that becomes available once thetrigger is cleaved attacks the carbonyl of the C(O)X-Therapeuticagent/drug (e.g., thereby leading to release of therapeutic agent-XH).function and thereby to quickly release the Drug-XH. In someembodiments, a driving force that permits the reaction to reachcompletion is the stability of the cyclic product. In some embodiments,a cyclization based linker of a dendrimer conjugate of the presentinvention include, but are not limited to, those shown in FIG. 23.

In some embodiments, a dendrimer conjugate of the present inventioncomprises a combination of one or more linkers. For example, in someembodiments, a dendrimer conjugate comprises a combination of two ormore elimination linkers. In some embodiments, a dendrimer conjugate ofthe present invention comprises two or more cyclization linkers. In someembodiments, a dendrimer conjugate of the present invention comprises aone or more elimination linkers and one or more cyclization linkers, ora combination of one or more different types of linkers describedherein. For example, in some embodiments, a dendrimer conjugatecomprises a linker as shown in FIG. 24.

In some embodiments, a dendrimer conjugate of the present inventioncomprises branched self-elimination linkers (e.g., as shown in FIG. 25).Thus, in some embodiments, use of branched linkers provides a conjugatethat can present increased concentrations of a therapeutic agent to atarget site (e.g., inflammatory site, tumor site, etc.).

In some embodiments, a dendrimer conjugate of the present invention isgenerated by a process comprising conjugating a pre-formed tripartitepiece (e.g., trigger, linker, and therapeutic agent) to a dendrimer(e.g., a G5 PAMAM dendrimer or other type of dendrimer described herein(e.g., conjugated to one or more different types of agents (e.g.,imaging agent)). In some embodiments, linkage between a tripartite pieceand a dendrimer comprises a non-cleavable bond (e.g., an ether or anamide bond (e.g., thereby decreasing unwanted activation of a triggerand/or degradation of a linker and/or release of therapeutic drug). Insome embodiments, a linker (e.g., linear or other type of linkerdescribed herein) is utilized to attach a tripartite moiety (e.g.,trigger, linker, and therapeutic agent) to a dendrimer (e.g., in orderto increase drug release, decrease steric hindrance, and/or increasestability of the dendrimer). For example, in some embodiments, thepresent invention provides a dendrimer conjugate as shown in FIG. 26A-B.

In some embodiments, a dendrimer conjugate of the present inventioncomprises a dendrimer conjugated to a linker (e.g., optionallyconjugated to a trigger) that is conjugated to a therapeutic agent. Insome embodiments, the dendrimer conjugate comprises a self-immolativeconnector between an ester bond (e.g., that is to be cleaved) and thetherapeutic agent (e.g., thereby enhancing drug release). For example,although a mechanism is not necessary to practice the present inventionand the present invention is not limited to any particular mechanism ofaction, in some embodiments, a dendrimer conjugate of the presentinvention comprising an ester linkage undergoes esterase catalyzedhydrolysis (e.g., as shown in FIG. 27 (e.g., G5 dendrimer comprising aself-degradable spacer and therapeutic agent)). Thus, in contrast to adendrimer comprising a simple ester (e.g., a dendrimer in the topportion of FIG. 27 wherein therapeutic agent release may or may notoccur, e.g., if x=NH), in some embodiments, the present inventionprovides a dendrimer conjugate comprising an elimination linker (e.g., a1, 6, elimination linker/spacer as shown in the bottom portion of FIG.27 (e.g., that permits complete hydrolysis of the linker (e.g., at atarget site))).

The present invention is not limited to any particular therapeutic agentthat is part of a dendrimer conjugate comprising a linker as describedherein. Indeed, a dendrimer conjugated comprising a linker may comprisenearly any therapeutic agent comprising a hydroxyl and/or amino group.In some embodiments, the therapeutic agent is an anti-cancer drug oragent. For example, in some embodiments, the therapeutic agent isdoxorubicin (or an analog thereof) or paclitaxel (or an analog thereof).In some embodiments, a dendrimer conjugate of the invention comprises atherapeutic agent comprising a single reactive group (e.g., at a primaryor secondary position). In some embodiments, a dendrimer conjugate ofthe present invention is synthesized utilizing a selectiveprotection/deprotection strategy if multiple functional groups arepresent within a therapeutic agent. In some embodiments, a dendrimerconjugate of the present invention provides the ability to deliver atherapeutic agent that, when not in the context of the dendrimerconjugate (e.g., in the absence of conjugation to a dendrimer (e.g., adendrimer comprising a linker and a trigger (e.g., configured to shieldand/or mask the therapeutic drug and/or prohibit release of thetherapeutic drug until the dendrimer reaches and reacts with a targetsite))) is toxic to a subject (e.g., that is too toxic to be utilized totreat a subject). Thus, in some embodiments, the present inventionprovides dendrimer conjugates comprising therapeutic agents that sufferfrom delivery issues and/or toxicity issues and/or non-specificityissues in the absence of being conjugated to a dendrimer conjugate. Forexample, in some embodiments, the present invention provides a dendrimerconjugate comprising a therapeutic agent comprising a compound of thecamptothecin family (e.g., IRINOTECAN). IRINOTECAN is a prodrug of10-hydroxycamptothecin (SN-38), which is 1000-fold more cytotoxic thanIRINOTECAN. It has been reported that the conversion of irinotecan tohydroxycamptothecin has very low efficiency. Thus, in some embodiments,the present invention provides a dendrimer conjugate comprisinghydroxycamptothecin (See, e.g., FIG. 28).

The present invention is not limited by the type of linkerconfiguration. In some embodiments, the linker is conjugated via a freeamino group via an amide linkage (e.g., formed from an active ester(e.g., the N-hydroxysuccinimide ester)). In some embodiments, an esterlinkage remains in the conjugate after conjugation. In some embodiments,linkage occurs through a lysine residue. In some embodiments,conjugation occurs through a short-acting, degradable linkage. Thepresent invention is not limited by the type of degradable linkageutilized. Indeed, a variety of linkages are contemplated to be useful inthe present invention including, but not limited to, physiologicallycleavable linkages including ester, carbonate ester, carbamate, sulfate,phosphate, acyloxyalkyl ether, acetal, and ketal linkages. In someembodiments, a dendrimer conjugate comprises a cleavable linkage presentin the linkage between the dendrimer and linker and/or targeting agentand/or therapeutic agent present therein (e.g., such that when cleaved,no portion of the linkage remains on the dendrimer). In someembodiments, a dendrimer conjugate comprises a cleavable linkage presentin the linker itself (e.g., such that when cleaved, a small portion ofthe linkage remains on the dendrimer).

The present invention is not limited to any particular targeting agent.In some embodiments, the targeting agent is a moiety that has affinityfor a tumor associated factor. For example, a number of targeting agentsare contemplated to be useful in the present invention including, butnot limited to, RGD sequences, low-density lipoprotein sequences, aNAALADase inhibitor, epidermal growth factor, and other agents that bindwith specificity to a target cell (e.g., a cancer cell)). In someembodiments, the targeting agent is an antibody, receptor ligand,hormone, vitamin, or antigen. However, the present invention is notlimited by the nature of the targeting agent. In some embodiments, theantibody is specific for a disease-specific antigen. In someembodiments, the disease-specific antigen comprises a tumor-specificantigen. In some embodiments, the receptor ligand includes, but is notlimited to, a ligand for CFTR, EGFR, estrogen receptor, FGR2, folatereceptor, IL-2 receptor, glycoprotein, or VEGFR. In some embodiments,the receptor ligand is folic acid.

The present invention is not limited to cancer and/or tumor targetingagents. Indeed, dendrimers of the present invention can be targeted(e.g., via a linker conjugated to the dendrimer wherein the linkercomprises a targeting agent) to a variety of target cells or tissues(e.g., to a biologically relevant environment) via conjugation to anappropriate targeting agent. For example, in some embodiments, thetargeting agent is a moiety that has affinity for an inflammatory factor(e.g., a cytokine or a cytokine receptor moiety (e.g., TNF-α receptor)).In some embodiments, the targeting agent is a sugar, peptide, antibodyor antibody fragment, hormone, hormone receptor, or the like.

In some embodiments of the present invention, the targeting agentincludes, but is not limited to an antibody, receptor ligand, hormone,vitamin, and antigen, however, the present invention is not limited bythe nature of the targeting agent. In some embodiments, the antibody isspecific for a disease-specific antigen. In some embodiments, thedisease-specific antigen comprises a tumor-specific antigen. In someembodiments, the receptor ligand includes, but is not limited to, aligand for CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2receptor, glycoprotein, and VEGFR. In some embodiments, the receptorligand is folic acid.

The present invention also provides a method of treating a disease(e.g., cancer, inflammatory disease, chronic pain, autoimmune disease,etc.) comprising administering to a subject suffering from orsusceptible to disease a therapeutically effective amount of acomposition comprising a dendrimer conjugate (e.g., comprising a linkerand/or trigger and a therapeutic agent) described herein. The presentinvention is not limited by the type of cancer treated using thecompositions and methods of the present invention. Indeed, a variety ofcancer can be treated including, but not limited to, prostate cancer,colon cancer, breast cancer, lung cancer and epithelial cancer.Similarly, the present invention is not limited by the type ofinflammatory disease and/or chronic pain treated using the compositionsof the present invention. Indeed, a variety of diseases can be treatedincluding, but not limited to, arthritis (e.g., osteoarthritis,rheumatoid arthritis, etc.), inflammatory bowel disease (e.g., colitis,Chrohn's disease, etc.), autoimmune disease (e.g., lupus erythematosus,multiple sclerosis, etc.), inflammatory pelvic disease, etc.

In some embodiments, the present invention also provides a kitcomprising a composition comprising dendrimer conjugate comprising alinker and/or trigger and a therapeutic agent. In some embodiments, thekit comprises a fluorescent agent or bioluminescent agent.

In some embodiments of the present invention, the therapeutic agentincludes, but is not limited to, a chemotherapeutic agent, ananti-oncogenic agent, an anti-angiogenic agent, a tumor suppressoragent, an anti-microbial agent, or an expression construct comprising anucleic acid encoding a therapeutic protein, although the presentinvention is not limited by the nature of the therapeutic agent. Infurther embodiments, the therapeutic agent is protected with aprotecting group selected from photo-labile, radio-labile, andenzyme-labile protecting groups. In some embodiments, thechemotherapeutic agent is selected from a group consisting of, but notlimited to, platinum complex, verapamil, podophylltoxin, carboplatin,procarbazine, mechloroethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, adriamycin,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, bleomycin, etoposide, tamoxifen, paclitaxel, taxol,transplatinum, 5-fluorouracil, vincristin, vinblastin, bisphosphonate(e.g., CB3717), chemotherapeutic agents with high affinity for folicacid receptors, ALIMTA (Eli Lilly), and methotrexate. In someembodiments, the anti-oncogenic agent comprises an antisense nucleicacid (e.g., RNA, molecule). In certain embodiments, the antisensenucleic acid comprises a sequence complementary to an RNA of anoncogene. In preferred embodiments, the oncogene includes, but is notlimited to, abl, Bcl-2, Bcl-xL, erb, fms, gsp, hst, jun, myc, neu, raf;ras, ret, src, or trk. In some embodiments, the nucleic acid encoding atherapeutic protein encodes a factor including, but not limited to, atumor suppressor, cytokine, receptor, inducer of apoptosis, ordifferentiating agent. In preferred embodiments, the tumor suppressorincludes, but is not limited to, BRCA1, BRCA2, C-CAM, p16, p21, p53,p73, Rb, and p27. In preferred embodiments, the cytokine includes, butis not limited to, GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, β-interferon,γ-interferon, and TNF. In preferred embodiments, the receptor includes,but is not limited to, CFTR, EGFR, estrogen receptor, IL-2 receptor, andVEGFR. In preferred embodiments, the inducer of apoptosis includes, butis not limited to, AdE1B, Bad, Bak, Bax, Bid, Bik, Bim, Harakid, andICE-CED3 protease. In some embodiments, the therapeutic agent comprisesa short-half life radioisotope.

In some embodiments of the present invention, the biological monitoringagent comprises an agent that measures an effect of a therapeutic agent(e.g., directly or indirectly measures a cellular factor or reactioninduced by a therapeutic agent), however, the present invention is notlimited by the nature of the biological monitoring agent. In someembodiments, the monitoring agent is capable of detecting (e.g.,measuring) apoptosis caused by the therapeutic agent.

In some embodiments of the present invention, the imaging agentcomprises a radioactive label including, but not limited to ¹⁴C, ³⁶Cl,⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ¹²⁵I, ¹³¹I, ¹¹¹Ln, ¹⁵²Eu, ⁵⁹Fe, ⁶⁷Ga, ³²P, ¹⁸⁶Re, ³⁵S,⁷⁵Se, Tc-99m, and ¹⁷⁵Yb. In some embodiments, the imaging agentcomprises a fluorescing entity. In a preferred embodiment, the imagingagent is fluorescein isothiocyanate or 6-TAMARA.

In some embodiments, dendrimer conjugates of the present invention areconfigured to treat disease. In preferred embodiments, dendrimerconjugates of the present invention are configured such that they arereadily cleared from the subject (e.g., so that there is little to nodetectable toxicity at efficacious doses). In some embodiments, thedisease is a neoplastic disease, selected from, but not limited to,leukemia, acute leukemia, acute lymphocytic leukemia, acute myelocyticleukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia, chronic leukemia, chronic myelocytic, (granulocytic)leukemia, chronic lymphocytic leukemia, Polycythemia vera, lymphoma,Hodgkin's disease, non-Hodgkin's disease, Multiple myeloma,Waldenstrom's macroglobulinemia, Heavy chain disease, solid tumors,sarcomas and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, and neuroblastomaretinoblastoma. In some embodiments, thedisease is an inflammatory disease selected from the group consistingof, but not limited to, eczema, inflammatory bowel disease, rheumatoidarthritis, asthma, psoriasis, ischemia/reperfusion injury, ulcerativecolitis and acute respiratory distress syndrome. In some embodiments,the disease is a viral disease selected from the group consisting of,but not limited to, viral disease caused by hepatitis B, hepatitis C,rotavirus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), human T-cell lymphotropic virustype I (HTLV-I), human T-cell lymphotropic virus type II (HTLV-II),AIDS, DNA viruses such as hepatitis type B and hepatitis type C virus;parvoviruses, such as adeno-associated virus and cytomegalovirus;papovaviruses such as papilloma virus, polyoma viruses, and SV40;adenoviruses; herpes viruses such as herpes simplex type I (HSV-I),herpes simplex type II (HSV-II), and Epstein-Barr virus; poxviruses,such as variola (smallpox) and vaccinia virus; and RNA viruses, such ashuman immunodeficiency virus type I (HIV-I), human immunodeficiencyvirus type II (HIV-II), human T-cell lymphotropic virus type I (HTLV-I),human T-cell lymphotropic virus type II (HTLV-II), influenza virus,measles virus, rabies virus, Sendai virus, picornaviruses such aspoliomyelitis virus, coxsackieviruses, rhinoviruses, reoviruses,togaviruses such as rubella virus (German measles) and Semliki forestvirus, arboviruses, and hepatitis type A virus.

The present invention is not limited by the type of therapeutic agentdelivered via a dendrimer of the present invention. For example, atherapeutic may be any agent selected from the group comprising, but notlimited to, a chemotherapeutic agent, an anti-oncogenic agent, ananti-angiogenic agent, a tumor suppressor agent, an anti-microbialagent, or an expression construct comprising a nucleic acid encoding atherapeutic protein. Illustrative examples of these types of agents aredescribed herein.

The dendrimers of the present invention find use in the detection andtreatment of a variety of cancers. Indeed, the present invention is notlimited by the type of cancer to be treated. Thus, in some embodiments,the present invention provides compositions comprising dendrimerconjugates for the targeting and identification of angiogenesisassociated with cancers (e.g., carcinomas). For example, in someembodiments, a dendrimer conjugate of the present invention furthercomprises a targeting agent (e.g., folic acid moiety) that associateswith high affinity to a targeting agent ligand (e.g., receptor) on acancer cell (e.g., carcinoma cells and/or solid tumor cells). In someembodiments, dendrimer conjugate and a targeting agent, that target andidentify cancer cells and/or angiogenesis associated with cancer,further comprise a therapeutic agent that inhibits angiogenesis therebytreating the cancer. In some embodiments, treatment with dendrimersconjugates and an anti-angiogenic agent are used in combination withother dendrimers of the present invention, with other chemotherapeutictreatments, and/or as a treatment following surgical removal of a tumoror cancerous tissue. In some embodiments, a targeting moiety (e.g.,folic acid or other targeting moiety described herein) posseses a highaffinity for ligands (e.g., receptors or other types of proteins ormolecules) present on cancer cell possessing such ligands therebypermiting the targeting, identification and treatment of disease (e.g.,cancer) with little to no toxicity to surrounding healthy cells andtissue.

Dendrimer conjugates of the present invention are not limited by thetype of anti-angiogenic agent used. Indeed, a variety of anti-angiogenicagents are contemplated to be useful in the compositions of the presentinvention including, but not limited to, Batimastat, Marimastat, AG3340,Neovastat, PEX, TIMP-1, -2, -3, -4, PAI-1, -2, uPA Ab, uPAR Ab,Amiloride, Minocycline, tetracyclines, steroids, cartilage-derived TIMP,αvβ3 Ab:LM609 and Vitaxin, RGD containing peptides, αvβ5 Ab, Endostatin,Angiostatin, aaAT, IFN-α, IFN-γ, IL-12, nitric oxide synthaseinhibitors, TSP-1, TNP-470, Combretastatin A4, Thalidomide, Linomide,IFN-α, PF-4, prolactin fragment, Suramin and analogues, PPS, distamycinA analogues, FGF-2 Ab, antisense-FGF-2, Protamine, SU5416, solubleFlt-1, dominant-negative Flk-1, VEGF receptor ribosymes, VEGF Ab,Aspirin, NS-398, 6-AT, 6A5BU, 7-DX, Genistein, Lavendustin A, Ang-2,batimastat, marimastat, anti-αvβ3 monoclonal antibody (LM609)thrombospondin-1 (TSP-1) Angiostatin, endostatin, TNP-470,Combretastatin A-4, Anti-VEGF antibodies, soluble Flk-1, Flt-1receptors, inhibitors of tyrosine kinase receptors, SU5416,heparin-binding growth factors, pentosan polysulfate, platelet-derivedendothelial cell growth factor/Thymidine phosphorylase (PD-ECGF/TP), cox(e.g., cox-1 an cox-2) inhibitors (e.g., Celebrex and Vioxx), DT385,Tissue inhibitor of metalloprotease (TIMP-1, TIMP-2), Zinc, Plasminogenactivator-inhibitor-1 (PAI-1), p53 Rb, Interleukin-10 Interleukin-12,Angiopoietin-2, Angiotensin, Angiotensin II (AT2 receptor), Caveolin-1,caveolin-2, Angiopoietin-2, Angiotensin, Angiotensin II (AT2 receptor),Caveolin-1, caveolin-2, Endostatin, Interferon-alpha, Isoflavones,Platelet factor-4, Prolactin (16 Kd fragment), Thrombospondin,Troponin-1, Bay 12-9566, AG3340, CGS 27023A, CGS 27023A, COL-3,(Neovastat), BMS-275291, Penicillamine, TNP-470 (fumagillin derivative),Squalamine, Combretastatin, Endostatin, Penicillamine, FarnesylTransferase Inhibitor (FTI), -L-778,123, -SCH66336, -R115777, anti-VEGFantibody, Thalidomide, SU5416, Ribozyme, Angiozyme, SU6668,PTK787/ZK22584, Interferon-alpha, Interferon-alpha, Suramin, Vitaxin,EMD121974, Penicillamine, Tetrathiomolybdate, Captopril, serine proteaseinhibitors, CAI, ABT-627, CM101/ZDO101, Interleukin-12, IM862,PNU-145156E, those described in U.S. Patent App. No. 20050123605, hereinincorporated by reference in its entirety, and fragments or portions ofthe above that retain anti-angiogenic (e.g., angiostatic or inhibitoryproperties).

In some embodiments, dendrimer conjugates and methods of using the sameof the present invention are used in treatment and/or monitoring duringcancer therapy. However, the systems and compositions of the presentinvention find use in the treatment and monitoring of a variety ofdisease states or other physiological conditions, and the presentinvention is not limited to use with any particular disease state orcondition. Other disease states that find particular use with thepresent invention include, but are not limited to, cardiovasculardisease, viral disease, inflammatory disease, and proliferativedisorders.

In some embodiments, the present invention provides a dendrimerconjugate comprising a linker and/or trigger and a therapeutic agentthat is acetylated (e.g., partially acetylated). In other embodiments,the present invention provides methods of manufacturing a dendrimerconjugate as described herein (e.g., in Examples 1-7).

Some embodiments of the present invention provide compositionscomprising dendrimer conjugates further comprising one or morefunctional groups, the functional groups including, but not limited to,therapeutic agents, biological monitoring components, biological imagingcomponents, targeting components, and components to identify thespecific signature of cellular abnormalities. As such, in someembodiments, a therapeutic dendrimer conjugate of the present inventionis made up of individual dendrimers, each with one or more functionalgroups being specifically conjugated with or covalently linked to thedendrimer.

The following discussion describes individual component parts of thedendrimer and methods of making and using the same in some embodimentsof the present invention. To illustrate the design and use of thesystems and compositions of the present invention, the discussionfocuses on specific embodiments of the use of the compositions in thetreatment and monitoring of cancer. These specific embodiments areintended only to illustrate certain preferred embodiments of the presentinvention and are not intended to limit the scope thereof.

In some embodiments, the release of a therapeutic agent is facilitatedby the therapeutic component being attached to a labile protectinggroup, such as, for example, cisplatin or methotrexate being attached toa photolabile protecting group that becomes released by laser lightdirected at cells emitting a color of fluorescence (e.g., in addition toand/or in place of target activated activation of a trigger component ofa dendrimer conjugate). In some embodiments, the therapeutic device alsomay have a component to monitor the response of the tumor to therapy.For example, where a therapeutic agent of the dendrimer inducesapoptosis of a target cell (e.g., a cancer cell (e.g., a prostate cancercell)), the caspase activity of the cells may be used to activate agreen fluorescence. This allows apoptotic cells to turn orange,(combination of red and green) while residual cells remain red. Anynormal cells that are induced to undergo apoptosis in collateral damagefluoresce green.

As is clear from the above example, the use of the compositions of thepresent invention facilitates non-intrusive sensing, signaling, andintervention for cancer and other diseases and conditions. Sincespecific protocols of molecular alterations in cancer cells areidentified using this technique, non-intrusive sensing through thedendrimers is achieved and may then be employed automatically againstvarious tumor phenotypes.

I. Dendrimers

In some embodiments, compositions of the present invention comprisedendrimers wherein the dendrimers. Dendrimeric polymers have beendescribed extensively (See, e.g., Tomalia, Advanced Materials 6:529(1994); Angew, Chem. Int. Ed. Engl., 29:138 (1990); incorporated hereinby reference in their entireties). Dendrimer polymers are synthesized asdefined spherical structures typically ranging from 1 to 20 nanometersin diameter. Methods for manufactureing a G5 PAMAM dendrimer with aprotected core is shown (FIGS. 1-5). In preferred embodiments, theprotected core diamine is NH2-CH2-CH2-NHPG. Molecular weight and thenumber of terminal groups increase exponentially as a function ofgeneration (the number of layers) of the polymer (See, e.g., FIG. 9).Different types of dendrimers can be synthesized based on the corestructure that initiates the polymerization process (See e.g., FIGS.1-5).

The dendrimer core structures dictate several characteristics of themolecule such as the overall shape, density and surface functionality(See, e.g., Tomalia et al., Chem. Int. Ed. Engl., 29:5305 (1990)).Spherical dendrimers can have ammonia as a trivalent initiator core orethylenediamine (EDA) as a tetravalent initiator core (See, e.g., FIG.9). Recently described rod-shaped dendrimers (See, e.g., Yin et al., J.Am. Chem. Soc., 120:2678 (1998)) use polyethyleneimine linear cores ofvarying lengths; the longer the core, the longer the rod. Dendriticmacromolecules are available commercially in kilogram quantities and areproduced under current good manufacturing processes (GMP) forbiotechnology applications.

Dendrimers may be characterized by a number of techniques including, butnot limited to, electrospray-ionization mass spectroscopy, ¹³C nuclearmagnetic resonance spectroscopy, ¹H nuclear magnetic resonancespectroscopy (See, e.g., Example 5, FIG. 10(A) and Example 7, FIG. 14),high performance liquid chromatography (See, e.g., Example 5, FIG.10(B); and Example 6, FIG. 13), size exclusion chromatography withmulti-angle laser light scattering (See, e.g., Example 4, FIG. 8),ultraviolet spectrophotometry (See, e.g., Example 8, FIG. 17), capillaryelectrophoresis and gel electrophoresis. These tests assure theuniformity of the polymer population and are important for monitoringquality control of dendrimer manufacture for GMP applications and invivo usage.

Numerous U.S. Patents describe methods and compositions for producingdendrimers. Examples of some of these patents are given below in orderto provide a description of some dendrimer compositions that may beuseful in the present invention, however it should be understood thatthese are merely illustrative examples and numerous other similardendrimer compositions could be used in the present invention.

U.S. Pat. No. 4,507,466, U.S. Pat. No. 4,558,120, U.S. Pat. No.4,568,737, and U.S. Pat. No. 4,587,329 each describe methods of makingdense star polymers with terminal densities greater than conventionalstar polymers. These polymers have greater/more uniform reactivity thanconventional star polymers, i.e. 3rd generation dense star polymers.These patents further describe the nature of the amidoamine dendrimersand the 3-dimensional molecular diameter of the dendrimers.

U.S. Pat. No. 4,631,337 describes hydrolytically stable polymers. U.S.Pat. No. 4,694,064 describes rod-shaped dendrimers. U.S. Pat. No.4,713,975 describes dense star polymers and their use to characterizesurfaces of viruses, bacteria and proteins including enzymes. Bridgeddense star polymers are described in U.S. Pat. No. 4,737,550. U.S. Pat.No. 4,857,599 and U.S. Pat. No. 4,871,779 describe dense star polymerson immobilized cores useful as ion-exchange resins, chelation resins andmethods of making such polymers.

U.S. Pat. No. 5,338,532 is directed to starburst conjugates ofdendrimer(s) in association with at least one unit of carriedagricultural, pharmaceutical or other material. This patent describesthe use of dendrimers to provide means of delivery of highconcentrations of carried materials per unit polymer, controlleddelivery, targeted delivery and/or multiple species such as e.g., drugsantibiotics, general and specific toxins, metal ions, radionuclides,signal generators, antibodies, interleukins, hormones, interferons,viruses, viral fragments, pesticides, and antimicrobials.

U.S. Pat. No. 6,471,968 describes a dendrimer complex comprisingcovalently linked first and second dendrimers, with the first dendrimercomprising a first agent and the second dendrimer comprising a secondagent, wherein the first dendrimer is different from the seconddendrimer, and where the first agent is different than the second agent.

Other useful dendrimer type compositions are described in U.S. Pat. No.5,387,617, U.S. Pat. No. 5,393,797, and U.S. Pat. No. 5,393,795 in whichdense star polymers are modified by capping with a hydrophobic groupcapable of providing a hydrophobic outer shell. U.S. Pat. No. 5,527,524discloses the use of amino terminated dendrimers in antibody conjugates.

The use of dendrimers as metal ion carriers is described in U.S. Pat.No. 5,560,929. U.S. Pat. No. 5,773,527 discloses non-crosslinkedpolybranched polymers having a comb-burst configuration and methods ofmaking the same. U.S. Pat. No. 5,631,329 describes a process to producepolybranched polymer of high molecular weight by forming a first set ofbranched polymers protected from branching; grafting to a core;deprotecting first set branched polymer, then forming a second set ofbranched polymers protected from branching and grafting to the corehaving the first set of branched polymers, etc.

U.S. Pat. No. 5,902,863 describes dendrimer networks containinglipophilic organosilicone and hydrophilic polyanicloamine nanscopicdomains. The networks are prepared from copolydendrimer precursorshaving PAMAM (hydrophilic) or polyproyleneimine interiors andorganosilicon outer layers. These dendrimers have a controllable size,shape and spatial distribution. They are hydrophobic dendrimers with anorganosilicon outer layer that can be used for specialty membrane,protective coating, composites containing organic organometallic orinorganic additives, skin patch delivery, absorbants, chromatographypersonal care products and agricultural products.

U.S. Pat. No. 5,795,582 describes the use of dendrimers as adjuvants forinfluenza antigen. Use of the dendrimers produces antibody titer levelswith reduced antigen dose. U.S. Pat. No. 5,898,005 and U.S. Pat. No.5,861,319 describe specific immunobinding assays for determiningconcentration of an analyte. U.S. Pat. No. 5,661,025 provides details ofa self-assembling polynucleotide delivery system comprising dendrimerpolycation to aid in delivery of nucleotides to target site. This patentprovides methods of introducing a polynucleotide into a eukaryotic cellin vitro comprising contacting the cell with a composition comprising apolynucleotide and a dendrimer polyeation non-covalently coupled to thepolynucleotide.

Dendrimer-antibody conjugates for use in in vitro diagnosticapplications has previously been demonstrated (See, e.g., Singh et al.,Clin. Chem., 40:1845 (1994)), for the production ofdendrimer-chelant-antibody constructs, and for the development ofboronated dendrimer-antibody conjugates (for neutron capture therapy);each of these latter compounds may be used as a cancer therapeutic (See,e.g., Wu et al., Bioorg. Med. Chem. Lett., 4:449 (1994); Wiener et al.,Magn. Reson. Med. 31:1 (1994); Barth et al., Bioconjugate Chem. 5:58(1994); and Barth et al.).

Some of these conjugates have also been employed in the magneticresonance imaging of tumors (See, e.g., Wu et al., (1994) and Wiener etal., (1994), supra). Results from this work have documented that, whenadministered in vivo, antibodies can direct dendrimer-associatedtherapeutic agents to antigen-bearing tumors. Dendrimers also have beenshown to specifically enter cells and carry either chemotherapeuticagents or genetic therapeutics. In particular, studies show thatcisplatin encapsulated in dendrimer polymers has increased efficacy andis less toxic than cisplatin delivered by other means (See, e.g., Duncanand Malik, Control Rel. Bioact. Mater. 23:105 (1996)).

Dendrimers have also been conjugated to fluorochromes or molecularbeacons and shown to enter cells. They can then be detected within thecell in a manner compatible with sensing apparatus for evaluation ofphysiologic changes within cells (See, e.g., Baker et al., Anal. Chem.69:990 (1997)). Finally, dendrimers have been constructed asdifferentiated block copolymers where the outer portions of the moleculemay be digested with either enzyme or light-induced catalysis (See,e.g., Urdea and Horn, Science 261:534 (1993)). This allows thecontrolled degradation of the polymer to release therapeutics at thedisease site and provides a mechanism for an external trigger to releasethe therapeutic agents.

II. Therapeutic Agents

A wide range of therapeutic agents find use with the present invention.Any therapeutic agent that can be associated with a dendrimer and/orlinker described herein may be delivered using the methods, systems, andcompositions of the present invention. For example, in some embodimentsthe therapeutic agent is Naloxone and/or a Naloxone pro-drug.

To illustrate delivery of therapeutic agents, the following discussionfocuses mainly on the delivery of methotrexate, cisplatin and taxol forthe treatment of cancer. Also discussed are various photodynamic therapycompounds. However, the present invention is not limited to solely tothe use of these exemplary agents. Indeed, a wide variety of agents(e.g., therapeutic agents) find use with the dendrimers of the presentinvention (e.g., as described herein).

i. Methotrexate, Cisplatin and Taxol

The cytotoxicity of methotrexate depends on the duration for which athreshold intracellular level is maintained (Levasseur et al., CancerRes 58, 5749 (1998); Goldman & Matherly, Pharmacol Ther 28, 77 (1985)).Cells contain high concentrations of DHFR, and, to shut off the DHFRactivity completely, anti-folate levels six orders of magnitude higherthan the Ki for DHFR is required (Sierrra & Goldman, Seminars inOncology 26, 11 (1999)). Furthermore, less than 5% of the enzymeactivity is sufficient for full cellular enzymatic function (White &Goldman, Biol Chem 256, 5722 (1981)). Cisplatin and Taxol have awell-defined action of inducing apoptosis in tumor cells (See e.g.,Lanni et al., Proc. Natl. Acad. Sci., 94:9679 (1997); Tortora et al.,Cancer Research 57:5107 (1997); and Zaffaroni et al., Brit. J. Cancer77:1378 (1998)). However, treatment with these and otherchemotherapeutic agents is difficult to accomplish without incurringsignificant toxicity. The agents currently in use are generally poorlywater soluble, quite toxic, and given at doses that affect normal cellsas wells as diseased cells. For example, paclitaxel (Taxol), one of themost promising anticancer compounds discovered, is poorly soluble inwater.

Paclitaxel has shown excellent antitumor activity in a wide variety oftumor models such as the B16 melanoma, L1210 leukemias, MX-1 mammarytumors, and CS-1 colon tumor xenografts. However, the poor aqueoussolubility of paclitaxel presents a problem for human administration.Accordingly, currently used paclitaxel formulations require a cremaphorto solubilize the drug. The human clinical dose range is 200-500 mg.This dose is dissolved in a 1:1 solution of ethanol:cremaphor anddiluted to one liter of fluid given intravenously. The cremaphorcurrently used is polyethoxylated castor oil. It is given by infusion bydissolving in the cremaphor mixture and diluting with large volumes ofan aqueous vehicle. Direct administration (e.g., subcutaneous) resultsin local toxicity and low levels of activity. Thus, there is a need formore efficient and effective delivery systems for these chemotherapeuticagents.

The present invention overcomes these problems by providing methods andcompositions for specific drug delivery. The present invention alsoprovides the ability to administer combinations of agents (e.g., two ormore different therapeutic agents) to produce an additive effect. Theuse of multiple agent may be used to counter disease resistance to anysingle agent. For example, resistance of some cancers to single drugs(taxol) has been reported (Yu et al., Molecular Cell. 2:581 (1998)). Insome embodiments, the present invention provides a dendrimer conjugatecomprising a linker conjugated to a chemotherapeutic agent (e.g., thetherapeutic agent methotrexate). In some embodiments, a dendrimerconjugate comprising a linker conjugated to methotrexate is used totarget and treat (e.g., kill) cancer cells (e.g., prostate cancer cells)within a subject. The present invention is contemplated to be useful fortreating a subject with any stage of cancer (e.g., prostate cancer). Insome embodiments, compositions of the present invention can be usedprophylactically.

The present invention also provides the opportunity to monitortherapeutic success following delivery of a therapeutic agent (e.g.,methotrexate, cisplatin and/or Taxol) to a subject. For example,measuring the ability of these drugs to induce apoptosis in vitro isreported to be a marker for in vivo efficacy (See, e.g., Gibb,Gynecologic Oncology 65:13 (1997)). Therefore, in addition to thetargeted delivery of a therapeutic agent (e.g., either one, two or allof the above mentioned drugs) to provide effective anti-tumor therapyand reduction of toxicity, the effectiveness of the therapy can begauged by a biological monitoring agent of the present invention (e.g.,that monitor the induction of apoptosis). It is contemplated thatdendrimer conjugates comprising a therapeutic agent and/or imagingagents and/or biological imaging agents are active against a wide-rangeof tumor types including, but not limited to, prostate cancer, breastcancer, colon cancer, lung cancer, epithelial cancer, etc.

Although the above discussion describes the specific therapeutic agentsmethotrexate, cisplatin and Taxol, any pharmaceutical that is routinelyused in a cancer therapy context finds use in the present invention. Intreating cancer according to the invention, the therapeutic component ofthe dendrimer may comprise compounds including, but not limited to,adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D,mitomycin C, or more preferably, cisplatin. The agent may be preparedand used as a combined therapeutic composition, or kit, by combining itwith an immunotherapeutic agent, as described herein.

In some embodiments of the present invention, a dendrimer conjugatecomprising linker is contemplated to comprise (e.g., be conjugated tovia a linker) one or more agents that directly cross-link nucleic acids(e.g., DNA) to facilitate DNA damage leading to a synergistic,antineoplastic agents of the present invention. Agents such ascisplatin, and other DNA alkylating agents may be used. Cisplatin hasbeen widely used to treat cancer, with efficacious doses used inclinical applications of 20 mg/M² for 5 days every three weeks for atotal of three courses. The dendrimers may be delivered via any suitablemethod, including, but not limited to, injection intravenously,subcutaneously, intratumorally, intraperitoneally, or topically (e.g.,to mucosal surfaces).

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 Mg/M²at 21 day intervals for adriamycin, to 35-50 Mg/M² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage and find use aschemotherapeutic agents in the present invention. A number of nucleicacid precursors have been developed. Particularly useful are agents thathave undergone extensive testing and are readily available. As such,agents such as 5-fluorouracil (5-FU) are preferentially used byneoplastic tissue, making this agent particularly useful for targetingto neoplastic cells. The doses delivered may range from 3 to 15mg/kg/day, although other doses may vary considerably according tovarious factors including stage of disease, amenability of the cells tothe therapy, amount of resistance to the agents and the like.

The anti-cancer therapeutic agents that find use in the presentinvention are those that are amenable to incorporation into dendrimerstructures or are otherwise associated with dendrimer structures suchthat they can be delivered into a subject, tissue, or cell without lossof fidelity of its anticancer effect. For a more detailed description ofcancer therapeutic agents such as a platinum complex, verapamil,podophyllotoxin, carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,bisulfan, nitrosurea, adriamycin, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastinand methotrexate and other similar anti-cancer agents, those of skill inthe art are referred to any number of instructive manuals including, butnot limited to, the Physician's Desk reference and to Goodman andGilman's “Pharmaceutical Basis of Therapeutics” ninth edition, Eds.Hardman et al., 1996.

In some embodiments, the drugs are preferably attached to the dendrimersand/or linkers conjugated to the dendrimers with photocleavable linkers.For example, several heterobifunctional, photocleavable linkers thatfind use with the present invention are described by Ottl et al. (Ottlet al., Bioconjugate Chem., 9:143 (1998)). These linkers can be eitherwater or organic soluble. They contain an activated ester that can reactwith amines or alcohols and an epoxide that can react with a thiolgroup. In between the two groups is a 3,4-dimethoxy6-nitrophenylphotoisomerization group, which, when exposed to near-ultraviolet light(365 nm), releases the amine or alcohol in intact form. Thus, thetherapeutic agent, when linked to the compositions of the presentinvention using such linkers, may be released in biologically active oractivatable form through exposure of the target area to near-ultravioletlight.

In some embodiments, methotrexate is conjugated to a dendrimer and/or toa linker conjugated to a dendrimer via an ester bond. In an exemplaryembodiment, the alcohol group of taxol is reacted with the activatedester of the organic-soluble linker. This product in turn is reactedwith the partially-thiolated surface of appropriate dendrimers (theprimary amines of the dendrimers can be partially converted tothiol-containing groups by reaction with a sub-stoichiometric amount of2-iminothiolano). In the case of cisplatin, the amino groups of the drugare reacted with the water-soluble form of the linker. If the aminogroups are not reactive enough, a primary amino-containing active analogof cisplatin, such as Pt(II) sulfadiazine dichloride (Pasani et al.,Inorg. Chim. Acta 80:99 (1983) and Abel et al, Eur. J. Cancer 9:4(1973)) can be used. Thus conjugated, the drug is inactive and will notharm normal cells. When the conjugate is localized within tumor cells,it is exposed to laser light of the appropriate near-UV wavelength,causing the active drug to be released into the cell.

Similarly, in other embodiments of the present invention, the aminogroups of cisplatin (or an analog thereof) is linked with a veryhydrophobic photocleavable protecting group, such as the2-nitrobenzyloxycarbonyl group (Pillai, V. N. R. Synthesis: 1-26(1980)). With this hydrophobic group attached, the drug is loaded intoand very preferentially retained by the hydrophobic cavities within thePAMAM dendrimer (See e.g., Esfand et al., Pharm. Sci., 2:157 (1996)),insulated from the aqueous environment. When exposed to near-LV light(about 365 nm), the hydrophobic group is cleaved, leaving the intactdrug. Since the drug itself is hydrophilic, it diffuses out of thedendrimer and into the tumor cell, where it initiates apoptosis.

An alternative to photocleavable linkers are enzyme cleavable linkers. Anumber of photocleavable linkers have been demonstrated as effectiveanti-tumor conjugates and can be prepared by attaching cancertherapeutics, such as doxorubicin, to water-soluble polymers withappropriate short peptide linkers (See e.g., Vasey et al., Clin. CancerRes., 5:83 (1999)). The linkers are stable outside of the cell, but arecleaved by thiolproteases once within the cell. In some embodiments, theconjugate PK1 is used. As an alternative to the photocleavable linkerstrategy, enzyme-degradable linkers, such as Gly-Phe-Leu-Gly may beused.

The present invention is not limited by the nature of the therapeutictechnique. For example, other conjugates that find use with the presentinvention include, but are not limited to, using conjugated borondusters for BNCT (Capala et al., Bioconjugate Chem., 7:7 (1996)), theuse of radioisotopes, and conjugation of toxins such as ricin to thedendrimer conjugate.

ii. Photodynamic Therapy

Photodynamic therapeutic agents may also be used as therapeutic agentsin the present invention. In some embodiments, the dendrimer conjugatesof the present invention containing photodynamic compounds areilluminated, resulting in the production of singlet oxygen and freeradicals that diffuse out of the fiberless radiative effector to act onthe biological target (e.g., tumor cells or bacterial cells). Somepreferred photodynamic compounds include, but are not limited to, thosethat can participate in a type H photochemical reaction:PS+hv

PS*(1)PS*(1)

PS*(3)PS*(3)+O₂

PS+*O₂*O₂+T

cytotoxitywhere PS=photosenstizer, PS*(1)=excited singlet state of PS,PS*(3)=excited triplet state of PS, hv=light quantum, *O₂=excitedsinglet state of oxygen, and T=biological target. Other photodynamiccompounds useful in the present invention include those that causecytotoxity by a different mechanism than singlet oxygen production(e.g., copper benzochlorin, Selman, et al., Photochem. Photobiol.,57:681-85 (1993), incorporated herein by reference). Examples ofphotodynamic compounds that find use in the present invention include,but are not limited to Photofrin 2, phtalocyanins (See e.g., Brasseur etal., Photochem. Photobiol., 47:705-11 (1988)), benzoporphyrin,tetrahydroxyphenylporphyrins, naphtalocyanines (See e.g., Firey andRodgers, Photochem. Photobiol., 45:535-38 (1987)), sapphyrins (See,e.g., Sessler et al., Proc. SPIE, 1426:318-29 (1991)), porphinones (See,e.g., Chang et al., Proc. SPIE, 1203:281-86 (1990)), tin etiopurpurin,ether substituted porphyrins (See, e.g., Pandey et al., Photochem.Photobiol., 53:65-72 (1991)), and cationic dyes such as the phenoxazines(See e.g., Cincotta et al., SPIE Proc., 1203:202-10 (1990)).III. Signature Identifying Agents

In some embodiments, dendrimer conjugates of the present inventioncontain one or more signature identifying agents that are activated by,or are able to interact with, a signature component (“signature”). Inpreferred embodiments, the signature identifying agent is an antibody,preferably a monoclonal antibody, that specifically binds the signature(e.g., cell surface molecule specific to a cell to be targeted).

In some embodiments of the present invention, tumor cells areidentified. Tumor cells have a wide variety of signatures, including thedefined expression of cancer-specific antigens such as Mucl, HER-2 andmutated p53 in breast cancer. These act as specific signatures for thecancer, being present in 30% (HER-2) to 70% (mutated p53) of breastcancers. In some embodiments, a dendrimer of the present inventioncomprises a monoclonal antibody that specifically binds to a mutatedversion of p53 that is present in breast cancer. In some embodiments, adendrimer of the present invention comprises an antibody (e.g.,monoclonal antibody) with high affinity for a signature including, butnot limited to, Mucl and HER-2.

In some embodiments of the present invention, cancer cells expressingsusceptibility genes are identified. For example, in some embodiments,there are two breast cancer susceptibility genes that are used asspecific signatures for breast cancer: BRCA1 on chromosome 17 and BRCA2on chromosome 13. When an individual carries a mutation in either BRCA1or BRCA2, they are at an increased risk of being diagnosed with breastor ovarian cancer at some point in their lives. These genes participatein repairing radiation-induced breaks in double-stranded DNA. It isthought that mutations in BRCA1 or BRCA2 might disable this mechanism,leading to more errors in DNA replication and ultimately to cancerousgrowth.

In addition, the expression of a number of different cell surfacereceptors find use as targets for the binding and uptake of a dendrimerconjugate. Such receptors include, but are not limited to, EGF receptor,folate receptor, FGR receptor 2, and the like.

In some embodiments of the present invention, changes in gene expressionassociated with chromosomal abborations are the signature component. Forexample, Burkitt lymphoma results from chromosome translocations thatinvolve the Myc gene. A chromosome translocation means that a chromosomeis broken, which allows it to associate with parts of other chromosomes.The classic chromosome translocation in Burkitt lymophoma involveschromosome 8, the site of the Myc gene. This changes the pattern of Mycexpression, thereby disrupting its usual function in controlling cellgrowth and proliferation.

In other embodiments, gene expression associated with colon cancer areidentified as the signature component. Two key genes are known to beinvolved in colon cancer: MSH2 on chromosome 2 and MLH1 on chromosome 3.Normally, the protein products of these genes help to repair mistakesmade in DNA replication. If the MSH2 and MLH1 proteins are mutated, themistakes in replication remain unrepaired, leading to damaged DNA andcolon cancer. MEN1 gene, involved in multiple endocrine neoplasia, hasbeen known for several years to be found on chromosome 11, was morefinely mapped in 1997, and serves as a signature for such cancers. Inpreferred embodiments of the present invention, an antibody specific forthe altered protein or for the expressed gene to be detected iscomplexed with nanodevices of the present invention.

In yet another embodiment, adenocarcinoma of the colon has definedexpression of CEA and mutated p53, both well-documented tumorsignatures. The mutations of p53 in some of these cell lines are similarto that observed in some of the breast cancer cells and allows for thesharing of a p53 sensing component between the two nanodevices for eachof these cancers (i.e., in assembling the nanodevice, dendrimerscomprising the same signature identifying agent may be used for eachcancer type). Both colon and breast cancer cells may be reliably studiedusing cell lines to produce tumors in nude mice, allowing foroptimization and characterization in animals.

From the discussion above it is clear that there are many differenttumor signatures that find use with the present invention, some of whichare specific to a particular type of cancer and others which arepromiscuous in their origin. The present invention is not limited to anyparticular tumor signature or any other disease-specific signature. Forexample, tumor suppressors that find use as signatures in the presentinvention include, but are not limited to, p53, Mucl, CEA, p16, p21,p27, CCAM, RB, APC, DCC, NF-1, NF-2, WT-1, MEN-1, MEN-H, p73, VHL, FCCand MCC.

IV. Biological Imaging Component

In some embodiments of the present invention, a dendrimer conjugatecomprises at least one imaging agent that can be readily imaged. Thepresent invention is not limited by the nature of the imaging componentused. In some embodiments of the present invention, imaging modulescomprise surface modifications of quantum dots (See e.g., Chan and Nie,Science 281:2016 (1998)) such as zinc sulfide-capped cadmium selenidecoupled to biomolecules (Sooklal, Adv. Mater., 10:1083 (1998)).

In some embodiments, the imaging module comprises dendrimers producedaccording to the “nanocomposite” concept (See, e.g., Balogh et al.,Proc. of ACS PMSE 77:118 (1997) and Balogh and Tomalia, J. Am. Che.Soc., 120:7355 (1998)). In these embodiments, dendrimers are produced byreactive encapsulation, where a reactant is preorganized by thedendrimer template and is then subsequently immobilized in/on thepolymer molecule by a second reactant. Size, shape, size distributionand surface functionality of these nanoparticles are determined andcontrolled by the dendritic macromolecules. These materials have thesolubility and compatibility of the host and have the optical orphysiological properties of the guest molecule (i.e., the molecule thatpermits imaging). While the dendrimer host may vary according to themedium, it is possible to load the dendrimer hosts with differentcompounds and at various guest concentration levels. Complexes andcomposites may involve the use of a variety of metals or other inorganicmaterials. The high electron density of these materials considerablysimplifies the imaging by electron microscopy and related scatteringtechniques. In addition, properties of inorganic atoms introduce new andmeasurable properties for imaging in either the presence or absence ofinterfering biological materials. In some embodiments of the presentinvention, encapsulation of gold, silver, cobalt, iron atoms/moleculesand/or organic dye molecules such as fluorescein are encapsulated intodendrimers for use as nanoscopi composite labels/tracers, although anymaterial that facilitates imaging or detection may be employed. In apreferred embodiment, the imaging agent is fluorescein isothiocyanate.

In some embodiments of the present invention, imaging is based on thepassive or active observation of local differences in density ofselected physical properties of the investigated complex matter. Thesedifferences may be due to a different shape (e.g., mass density detectedby atomic force microscopy), altered composition (e.g. radiopaquesdetected by X-ray), distinct light emission (e.g., fluorochromesdetected by spectrophotometry), different diffraction (e.g.,electron-beam detected by TEM), contrasted absorption (e.g., lightdetected by optical methods), or special radiation emission (e.g.,isotope methods), etc. Thus, quality and sensitivity of imaging dependon the property observed and on the technique used. The imagingtechniques for cancerous cells have to provide sufficient levels ofsensitivity to is observe small, local concentrations of selected cells.The earliest identification of cancer signatures requires highselectivity (i.e., highly specific recognition provided by appropriatetargeting) and the highest possible sensitivity.

A. Magnetic Resonance Imaging

In some embodiments, once a targeted dendrimer conjugate has attached to(or been internalized into) a target cell (e.g., tumor cell and orinflammatory cell), one or more modules on the device serve to image itslocation. Dendrimers have already been employed as biomedical imagingagents, perhaps most notably for magnetic resonance imaging (MRI)contrast enhancement agents (See e.g., Wiener et al., Mag. Reson. Med.31:1 (1994); an example using PAMAM dendrimers). These agents aretypically constructed by conjugating chelated paramagnetic ions, such asGd(III)-diethylenetriaminepentaacetic acid (Gd(III)-DTPA), towater-soluble dendrimers. Other paramagnetic ions that may be useful inthis context include, but are not limited to, gadolinium, manganese,copper, chromium, iron, cobalt, erbium, nickel, europium, technetium,indium, samarium, dysprosium, ruthenium, ytterbium, yttrium, and holmiumions and combinations thereof. In some embodiments of the presentinvention, a dendrimer conjugate is also conjugated to a targetinggroup, such as epidermal growth factor (EGF), to make the conjugatespecifically bind to the desired cell type (e.g., in the case of EGF,EGFR-expressing tumor cells). In a preferred embodiment of the presentinvention, DTPA is attached to dendrimers via the isothiocyanate of DTPAas described by Wiener (Wiener et al., Mag. Reson. Med. 31:1 (1994)).

Dendrimeric MRI agents are particularly effective due to thepolyvalency, size and architecture of dendrimers, which results inmolecules with large proton relaxation enhancements, high molecularrelaxivity, and a high effective concentration of paramagnetic ions atthe target site. Dendrimeric gadolinium contrast agents have even beenused to differentiate between benign and malignant breast tumors usingdynamic MRI, based on how the vasculature for the latter type of tumorimages more densely (Adam et al., Ivest. Rad. 31:26 (1996)). Thus, MRIprovides a particularly useful imaging system of the present invention.

B. Microscopic Imaging

Static structural microscopic imaging of cancerous cells and tissues hastraditionally been performed outside of the patient. Classical histologyof tissue biopsies provides a fine illustrative example, and has provena powerful adjunct to cancer diagnosis and treatment. After removal, aspecimen is sliced thin (e.g., less than 40 microns), stained, fixed,and examined by a pathologist. If images are obtained, they are mostoften 2-D transmission bright-field projection images. Specialized dyesare employed to provide selective contrast, which is almost absent fromthe unstained tissue, and to also provide for the identification ofaberrant cellular constituents. Quantifying sub-cellular structuralfeatures by using computer-assisted analysis, such as in nuclear ploidydetermination, is often confounded by the loss of histologic contextowing to the thinness of the specimen and the overall lack of 3-Dinformation. Despite the limitations of the static imaging approach, ithas been invaluable to allow for the identification of neoplasia inbiopsied tissue. Furthermore, its use is often the crucial factor in thedecision to perform invasive and risky combinations of chemotherapy,surgical procedures, and radiation treatments, which are oftenaccompanied by severe collateral tissue damage, complications, and evenpatient death.

A dendrimer conjugate of the present invention allows functionalmicroscopic imaging of tumors and provide improved methods for imaging.The methods find use in vivo, in vitro, and ex vivo. For example, in oneembodiment of the present invention, dendrimer conjugates of the presentinvention are designed to emit light or other detectable signals uponexposure to light. Although the labeled dendrimers may be physicallysmaller than the optical resolution limit of the microscopy technique,they become self-luminous objects when excited and are readilyobservable and measurable using optical techniques. In some embodimentsof the present invention, sensing fluorescent biosensors in a microscopeinvolves the use of tunable excitation and emission filters andmultiwavelength sources (See, e.g., Farkas et al., SPEI 2678:200(1997)). In embodiments where the imaging agents are present in deepertissue, longer wavelengths in the Near-infrared (NMR) are used (Seee.g., Lester et al., Cell Mol. Biol. 44:29 (1998)). Dendrimericbiosensing in the Near-IR has been demonstrated with dendrimericbiosensing antenna-like architectures (See, e.g., Shortreed et al., J.Phys. Chem., 101:6318 (1997)). Biosensors that find use with the presentinvention include, but are not limited to, fluorescent dyes andmolecular beacons.

In some embodiments of the present invention, in vivo imaging isaccomplished using functional imaging techniques. Functional imaging isa complementary and potentially more powerful techniques as compared tostatic structural imaging. Functional imaging is best known for itsapplication at the macroscopic scale, with examples including functionalMagnetic Resonance Imaging (fMRI) and Positron Emission Tomography(PET). However, functional microscopic imaging may also be conducted andfind use in in vivo and ex vivo analysis of living tissue. Functionalmicroscopic imaging is an efficient combination of 3-D imaging, 3-Dspatial multispectral volumetric assignment, and temporal sampling: inshort a type of 3-D spectral microscopic movie loop. Interestingly,cells and tissues autofluoresce. When excited by several wavelengths,providing much of the basic 3-D structure needed to characterize severalcellular components (e.g., the nucleus) without specific labeling.Oblique light illumination is also useful to collect structuralinformation and is used routinely. As opposed to structural spectralmicroimaging, functional spectral microimaging may be used withbiosensors, which act to localize physiologic signals within the cell ortissue. For example, in some embodiments of the present invention,biosensor-comprising dendrimers of the present invention are used toimage upregulated receptor families such as the folate or EGF classes.In such embodiments, functional biosensing therefore involves thedetection of physiological abnormalities relevant to carcinogenesis ormalignancy, even at early stages. A number of physiological conditionsmay be imaged using the compositions and methods of the presentinvention including, but not limited to, detection of nanoscopicdendrimeric biosensors for pH, oxygen concentration, Ca²+ concentration,and other physiologically relevant analytes.

V. Biological Monitoring Component

The biological monitoring or sensing component of a dendrimer conjugateof the present invention is one that can monitor the particular responsein a target cell (e.g., tumor cell) induced by an agent (e.g., atherapeutic agent provided by the therapeutic component of the dendrimerconjugate). While the present invention is not limited to any particularmonitoring system, the invention is illustrated by methods andcompositions for monitoring cancer treatments. In preferred embodimentsof the present invention, the agent induces apoptosis in cells andmonitoring involves the detection of apoptosis. In particularembodiments, the monitoring component is an agent that fluoresces at aparticular wavelength when apoptosis occurs. For example, in a preferredembodiment, caspase activity activates green fluorescence in themonitoring component. Apoptotic cancer cells, which have turned red as aresult of being targeted by a particular signature with a red label,turn orange while residual cancer cells remain red. Normal cells inducedto undergo apoptosis (e.g., through collateral damage), if present, willfluoresce green.

In these embodiments, fluorescent groups such as fluorescein areemployed in the monitoring component. Fluorescein is easily attached tothe dendrimer surface via the isothiocyanate derivatives, available fromMOLECULAR PROBES, Inc. This allows the dendrimer conjugate to be imagedwith the cells via confocal microscopy. Sensing of the effectiveness ofthe dendrimer conjugates is preferably achieved by using fluorogenicpeptide enzyme substrates. For example, apoptosis caused by thetherapeutic agents results in the production of the peptidase caspase-1(ICE). CALBIOCHEM sells a number of peptide substrates for this enzymethat release a fluorescent moiety. A particularly useful peptide for usein the present invention is:

(SEQ ID NO: 1) MCA-Tyr-Glu-Val-Asp-Gly-Trp-Lys-(DNP)-NH₂where MCA is the (7-methoxycoumarin-4-yl)acetyl and DNP is the2,4-dinitrophenyl group (See, e.g., Talanian et al., J. Biol. Chem.,272: 9677 (1997)). In this peptide, the MCA group has greatly attenuatedfluorescence, due to fluorogenic resonance energy transfer (FRET) to theDNP group. When the enzyme cleaves the peptide between the aspartic acidand glycine residues, the MCA and DNP are separated, and the MCA groupstrongly fluoresces green (excitation maximum at 325 nm and emissionmaximum at 392 nm).

In some embodiments of the present invention, the lysine end of thepeptide is linked to the dendrimer conjugate, so that the MCA group isreleased into the cytosol when it is cleaved. The lysine end of thepeptide is a useful synthetic handle for conjugation because, forexample, it can react with the activated ester group of a bifunctionallinker such as Mal-PEG-OSu. Thus the appearance of green fluorescence inthe target cells produced using these methods provides a clearindication that apoptosis has begun (if the cell already has a red colorfrom the presence of aggregated quantum dots, the cell turns orange fromthe combined colors).

Additional fluorescent dyes that find use with the present inventioninclude, but are not limited to, acridine orange, reported as sensitiveto DNA changes in apoptotic cells (Abrams et al., Development 117:29(1993)) and cis-parinaric acid, sensitive to the lipid peroxidation thataccompanies apoptosis (Hockenbery et al., Cell 75:241 (1993)). It shouldbe noted that the peptide and the fluorescent dyes are merely exemplary.It is contemplated that any peptide that effectively acts as a substratefor a caspase produced as a result of apoptosis finds use with thepresent invention.

VI. Targeting Agents

As described above, another component of the present invention is thatthe dendrimer conjugate compositions are able to specifically target aparticular cell type (e.g., tumor cell). In some embodiments, thedendrimer conjugate targets neoplastic cells through a cell surfacemoiety and is taken into the cell through receptor mediated endocytosis.

In some embodiments of the present invention, targeting groups areconjugated to dendrimers and/or linkers conjugated to the dendrimerswith either short (e.g., direct coupling), medium (e.g. usingsmall-molecule bifunctional linkers such as SPDP, sold by PIERCECHEMICAL Company), or long (e.g., PEG bifunctional linkers, sold byNEKTAR, Inc.) linkages. Since dendrimers have surfaces with a largenumber of functional groups, more than one targeting group and/or linkermay be attached to each dendrimer. As a result, multiple binding eventsmay occur between the dendrimer conjugate and the target cell. In theseembodiments, the dendrimer conjugates have a very high affinity fortheir target cells via this “cooperative binding” or polyvalentinteraction effect.

For steric reasons, in some embodiments, the smaller the ligands, themore can be attached to the surface of a dendrimer and/or linkersattached thereto. Recently, Wiener reported that dendrimers withattached folic acid would specifically accumulate on the surface andwithin tumor cells expressing the high-affinity folate receptor (hFR)(See, e.g., Wiener et al., Invest. Radiol., 32:748 (1997)). The hFRreceptor is expressed or upregulated on epithelial tumors, includingbreast cancers. Control cells lacking hFR showed no significantaccumulation of folate-derivatized dendrimers. Folic acid can beattached to full generation PAMAM dendrimers via a carbodiimide couplingreaction. Folic acid is a good targeting candidate for the dendrimers,with its small size and a simple conjugation procedure.

Antibodies can be generated to allow for the targeting of antigens orimmunogens (e.g., tumor, tissue or pathogen specific antigens) onvarious biological targets (e.g., pathogens, tumor cells, normaltissue). Such antibodies include, but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary.

In some embodiments, the antibodies recognize tumor specific epitopes(e.g., TAG-72 (See, e.g., Kjeldsen et al., Cancer Res. 48:2214-2220(1988); U.S. Pat. Nos. 5,892,020; 5,892,019; and 5,512,443); humancarcinoma antigen (See, e.g., U.S. Pat. Nos. 5,693,763; 5,545,530; and5,808,005); TP1 and TP3 antigens from osteocarcinoma cells (See, e.g.,U.S. Pat. No. 5,855,866); Thomsen-Friedenreich (TF) antigen fromadenocarcinoma cells (See, e.g., U.S. Pat. No. 5,110,911); “KC-4antigen” from human prostrate adenocarcinoma (See, e.g., U.S. Pat. Nos.4,708,930 and 4,743,543); a human colorectal cancer antigen (See, e.g.,U.S. Pat. No. 4,921,789); CA125 antigen from cystadenocarcinoma (See,e.g., U.S. Pat. No. 4,921,790); DF3 antigen from human breast carcinoma(See, e.g., U.S. Pat. Nos. 4,963,484 and 5,053,489); a human breasttumor antigen (See, e.g., U.S. Pat. No. 4,939,240); p97 antigen of humanmelanoma (See, e.g., U.S. Pat. No. 4,918,164); carcinoma ororosomucoid-related antigen (CORA) (See, e.g., U.S. Pat. No. 4,914,021);a human pulmonary carcinoma antigen that reacts with human squamous celllung carcinoma but not with human small cell lung carcinoma (See, e.g.,U.S. Pat. No. 4,892,935); T and Tn haptens in glycoproteins of humanbreast carcinoma (See, e.g., Springer et al., Carbohydr. Res.178:271-292 (1988)), MSA breast carcinoma glycoprotein termed (See,e.g., Tjandra et al., Br. J. Surg. 75:811-817 (1988)); MFGM breastcarcinoma antigen (See, e.g., Ishida et al., Tumor Biol. 10:12-22(1989)); DU-PAN-2 pancreatic carcinoma antigen (See, e.g., Lan et al.,Cancer Res. 45:305-310 (1985)); CA125 ovarian carcinoma antigen (See,e.g., Hanisch et al., Carbohydr. Res. 178:29-47 (1988)); YH206 lungcarcinoma antigen (See, e.g., Hinoda et al., (1988) Cancer J. 42:653-658(1988)). Each of the foregoing references are specifically incorporatedherein by reference.

Various procedures known in the art are used for the production ofpolyclonal antibodies. For the production of antibody, various hostanimals can be immunized by injection with the peptide corresponding tothe desired epitope including but not limited to rabbits, mice, rats,sheep, goats, etc. In a preferred embodiment, the peptide is conjugatedto an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants are usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (See e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Kohler and Milstein (Kohler and Milstein, Nature256:495-497 (1975)), as well as the trioma technique, the human B-cellhybridoma technique (See e.g., Kozbor et al. Immunol. Today 4:72(1983)), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 (1985)).

In an additional embodiment of the invention, monoclonal antibodies canbe produced in germ-free animals utilizing recent technology (See e.g.,PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., Proc.Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)) or by transforming human Bcells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, pp. 77-96 (1985)).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies.An additional embodiment of the invention utilizes the techniquesdescribed for the construction of Fab expression libraries (Huse et al.,Science 246:1275-1281 (1989)) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)2 fragment thatcan be produced by pepsin digestion of the antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and the Fab fragments that can be generated bytreating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art (e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.).

The dendrimer conjugates of the present invention have many advantagesover liposomes, such as their greater stability, better control of theirsize and polydispersity, and generally lower toxicity and immunogenicity(See e.g., Duncan et al, Polymer Preprints 39:180 (1998)). Thus, in someembodiments of the present invention, anti-HER2 antibody fragments, aswell as other targeting antibodies are conjugated to dendrimers, astargeting agents for the nanodevices of the present invention.

The bifunctional linkers SPDP and SMCC and the longer Mal-PEG-OSulinkers are particularly useful for antibody-dendrimer conjugation. Inaddition, many tumor cells contain surface lectins that bind tooligosaccharides, with specific recognition arising chiefly from theterminal carbohydrate residues of the latter (See, e.g., Sharon and Lis, Science 246:227 (1989)). Attaching appropriate monosaccharides tononglycosylated proteins such as BSA provides a conjugate that binds totumor lectin much more tightly than the free monosaccharide (See, e.g.,Monsigny et al., Biochemie 70:1633 (1988)).

Mannosylated PAMAM dendrimers bind mannoside-binding lectin up to 400more avidly than monomeric mannosides (See, e.g., Page and Roy,Bioconjugate Chem., 8:714 (1997)). Sialylated dendrimers and otherdendritic polymers bind to and inhibit a variety of sialate-bindingviruses both in vitro and in vivo. By conjugating multiplemonosaccharide residues (e.g., α-galactoside, for galactose-bindingcells) to dendrimers, polyvalent conjugates are created with a highaffinity for the corresponding type of tumor cell. The attachmentreaction are easily carried out via reaction of the terminal amines withcommercially-available α-galactosidyl-phenylisothiocyanate. The smallsize of the carbohydrates allows a high concentration to be present onthe dendrimer surface.

Related to the targeting approaches described above is the“pretargeting” approach (See e.g., Goodwin and Meares, Cancer (suppl.)80:2675 (1997)). An example of this strategy involves initial treatmentof a subject with conjugates of tumor-specific monoclonal antibodies andstreptavidin. Remaining soluble conjugate is removed from thebloodstream with an appropriate biotinylated clearing agent. When thetumor-localized conjugate is all that remains, a radiolabeled,biotinylated agent is introduced, which in turn localizes at the tumorsites by the strong and specific biotin-streptavidin interaction. Thus,the radioactive dose is maximized in dose proximity to the cancer cellsand minimized in the rest of the body where it can harm healthy cells.

It has been shown that if streptavidin molecules bound to a polystyrenewell are first treated with a biotinylated dendrimer, and thenradiolabeled streptavidinis introduced, up to four of the labeledstreptavidin molecules are bound per polystyrene-bound streptavidin(See, e.g., Wilbur et al., Bioconjugate Chem., 9:813 (1998)). Thus,biotinylated dendrimers may be used in the methods of the presentinvention, acting as a polyvalent receptor for the radiolabel in vivo,with a resulting amplification of the radioactive dosage per boundantibody conjugate. In the preferred embodiments of the presentinvention, one or more multiply-biotinylated module(s) on the clustereddendrimer presents a polyvalent target for radiolabeled or boronated(See, e.g., Barth et al., Cancer Investigation 14:534 (1996)) avidin orstreptavidin, again resulting in an amplified dose of radiation for thetumor cells.

Dendrimers may also be used as clearing agents by, for example,partially biotinylating a dendrimer that has a polyvalent galactose ormannose surface. The conjugate-clearing agent complex would then have avery strong affinity for the corresponding hepatocyte receptors.

In other embodiments of the present invention, an enhanced permeabilityand retention (EPR) method is used in targeting. The enhancedpermeability and retention (EPR) effect is a more “passive” way oftargeting tumors (See, e.g., Duncan and Sat, Ann. Oncol., 9:39 (1998)).The EPR effect is the selective concentration of macromolecules andsmall particles in the tumor microenvironment, caused by thehyperpermeable vasculature and poor lymphatic drainage of tumors. Thedendrimer compositions of the present invention provide ideal polymersfor this application, in that they are relatively rigid, of narrowpolydispersity, of controlled size and surface chemistry, and haveinterior “cargo” space that can carry and then release antitumor drugs.In fact, PAMAM dendrimer-platinates have been shown to accumulate insolid tumors (Pt levels about 50 times higher than those obtained withcisplatin) and have in vivo activity in solid tumor models for whichcisplatin has no effect (See, e.g., Malik et al., Proc. Intl. Symp.Control. Rel. Bioact. Mater., 24:107 (1997) and Duncan et al., PolymerPreprints 39:180 (1998)).

VII. Synthesis and Conjugation

The present section provides a description of the synthesis andformation of dendrimer conjugates described above (See, e.g., Examples1-7).

In some embodiments of the present invention, the preparation of PAMAMdendrimers is performed according to a typical divergent (building upthe macromolecule from an initiator core) synthesis. It involves atwo-step growth sequence that includes of a Michael addition of aminogroups to the double bond of methyl acrylate (MA) followed by theamidation of the resulting terminal carbomethoxy, —(CO₂ CH₃) group, withethylenediamine (EDA).

In the first step of this process, ammonia is allowed to react under aninert nitrogen atmosphere with MA (molar ratio: 1:4.25) at 47° C. for 48hours. The resulting compound is referred to as generation=0, thestar-branched PAMAM tri-ester. The next step involves reacting thetri-ester with an excess of EDA to produce the star-branched PAMAMtri-amine (G=O). This reaction is performed under an inert atmosphere(nitrogen) in methanol and requires 48 hours at 0° C. for completion.Reiteration of this Michael addition and amidation sequence producesgeneration=1.

Preparation of this tri-Amine Completes the First Full Cycle of theDivergent Synthesis of PAMAM dendrimers. Repetition of this reactionsequence results in the synthesis of larger generation (G=1-5)dendrimers (i.e., ester- and amine-terminated molecules, respectively).For example, the second iteration of this sequence produces generation1, with an hexa-ester and hexa-amine surface, respectively. The samereactions are performed in the same way as for all subsequentgenerations from 1 to 9, building up layers of branch cells giving acore-shell architecture with precise molecular weights and numbers ofterminal groups as shown above. Carboxylate-surfaced dendrimers can beproduced by hydrolysis of ester-terminated PAMAM dendrimers, or reactionof succinic anhydride with amine-surfaced dendrimers (e.g., fullgeneration PAMAM, POPAM or POPAM-PAMAM hybrid dendrimers).

Various dendrimers can be synthesized based on the core structure thatinitiates the polymerization process. These core structures dictateseveral important characteristics of the dendrimer molecule such as theoverall shape, density, and surface functionality (See, e.g., Tomalia etal., Angew. Chem. Int. Ed. Engl., 29:5305 (1990)). Spherical dendrimersderived from ammonia possess trivalent initiator cores, whereas EDA is atetra-valent initiator core. Recently, rod-shaped dendrimers have beenreported which are based upon linear poly(ethyleneimine) cores ofvarying lengths the longer the core, the longer the rod (See, e.g., Yinet al., J. Am. Chem. Soc., 120:2678 (1998)).

In some embodiments, dendrimers of the present invention comprise aprotected core diamine. In some embodiments, the protected initiatorcore diamine is NH2-(CH2)_(n)-NHPG, (n=1-10). In other embodiments, theintitor core is selected from the group comprising, but not limited to,NH2-(CH2)_(n)-NH2 (n=1-10), NH2-((CH2)_(n)NH2)₃ (n=1-10), orunsubstituted or substituted 1,2-; 1,3-; or1,4-phenylenedi-n-alkylamine, with a monoprotected diamine (e.g.,NH2-(CH2)_(n)-NHPG) used during the amide formation of each generation.In these approaches, the protected diamine allows for the large scaleproduction of dendrimers without the production of non-uniformnanostructures that can make characterization and analysis difficult. Bylimiting the reactivity of the diamine to only one terminus, theopportunities of dimmer/polymer formation and intramolecular reactionsare obviated without the need of employing large excesses of diamine.The terminus monoprotected intermediates can be readily purified sincethe protecting groups provide suitable handle for productivepurifications by classical techniques like crystallization and orchromatography.

The protected intermediates can be deprotected in a deprotection step,and the resulting generation of the dendrimer subjected to the nextiterative chemical reaction without the need for purification. Theinvention is not limited to a particular protecting group. Indeed avariety of protecting groups are contemplated including, but not limitedto, t-butoxycarbamate (N-t-Boc), allyloxycarbamate (N-Alloc),benzylcarbamate (N-Cbz), 9-fluorenylmethylcarbamate (FMOC), orphthalimide (Phth). In preferred embodiments of the present invention,the protecting group is benzylcarbamate (N-Cbz). N-Cbz is ideal for thepresent invention since it alone can be easily cleaved under “neutral”conditions by catalytic hydrogenation (Pd/C) without resorting tostrongly acidic or basic conditions needed to remove an F-MOC group. Theuse of protected monomers finds particular use in high through-putproduction runs because a lower amount of monomer can be used, reducingproduction costs.

The dendrimers may be characterized for size and uniformity by anysuitable analytical techniques. These include, but are not limited to,atomic force microscopy (AFM), electrospray-ionization massspectroscopy, MALDI-TOF mass spectroscopy, ¹³C nuclear magneticresonance spectroscopy, high performance liquid chromatography (HPLC)size exclusion chromatography (SEC) (equipped with multi-angle laserlight scattering, dual UV and refractive index detectors), capillaryelectrophoresis and get electrophoresis. These analytical methods assurethe uniformity of the dendrimer population and are important in thequality control of dendrimer production for eventual use in in vivoapplications. Most importantly, extensive work has been performed withdendrimers showing no evidence of toxicity when administeredintravenously (Roberts et al., J. Biomed. Mater. Res., 30:53 (1996) andBoume et al., J. Magnetic Resonance Imaging, 6:305 (1996)).

VIII. Evaluation of Anti-Tumor Efficacy and Toxicity of Dendrimers

The anti-tumor effects of various therapeutic agents on cancer celllines and primary cell cultures may be evaluated using the nanodevicesof the present invention. For example, in preferred embodiments, assaysare conducted, in vitro, using established tumor cell line models orprimary culture cells.

A. Quantifying the Induction of Apoptosis of Human Tumor Cells In Vitro

In an exemplary embodiment of the present invention, the dendrimerconjugates of the present invention are used to assay apoptosis of humantumor cells in vitro. Testing for apoptosis in the cells determines theefficacy of the therapeutic agent. Multiple aspects of apoptosis can andshould be measured. These aspects include those described above, as wellas aspects including, but are not limited to, measurement ofphosphatidylserine (PS) translocation from the inner to outer surface ofplasma membrane, measurement of DNA fragmentation, detection ofapoptosis related proteins, and measurement of Caspase-3 activity.

B. In Vitro Toxicology

In some embodiments of the present invention, to gain a generalperspective into the safety of a particular dendrimer conjugate platformor component of that system, toxicity testing is performed.Toxicological information may be derived from numerous sourcesincluding, but not limited to, historical databases, in vitro testing,and in vivo animal studies.

In vitro toxicological methods have gained popularity in recent yearsdue to increasing desires for alternatives to animal experimentation andan increased perception to the potential ethical, commercial, andscientific value. In vitro toxicity testing systems have numerousadvantages including improved efficiency, reduced cost, and reducedvariability between experiments. These systems also reduce animal usage,eliminate confounding systemic effects (e.g., immunity), and controlenvironmental conditions.

Although any in vitro testing system may be used with the presentinvention, the most common approach utilized for in vitro examination isthe use of cultured cell models. These systems include freshly isolatedcells, primary cells, or transformed cell cultures. Cell culture as theprimary means of studying in vitro toxicology is advantageous due torapid screening of multiple cultures, usefulness in identifying andassessing toxic effects at the cellular, subcellular, or molecularlevel. In vitro cell culture methods commonly indicate basic cellulartoxicity through measurement of membrane integrity, metabolicactivities, and subcellular perturbations. Commonly used indicators formembrane integrity include cell viability (cell count), clonal expansiontests, trypan blue exclusion, intracellular enzyme release (e.g. lactatedehydrogenase), membrane permeability of small ions (K¹, Ca²⁺), andintracellular Ala accumulation of small molecules (e.g., ⁵¹Cr,succinate). Subcellular perturbations include monitoring mitochondrialenzyme activity levels via, for example, the MTT test, determiningcellular adenine triphosphate (ATP) levels, neutral red uptake intolysosomes, and quantification of total protein synthesis. Metabolicactivity indicators include glutathione content, lipid peroxidation, andlactate/pyruvate ratio.

C. MTT Assay

The MTT assay is a fast, accurate, and reliable methodology forobtaining cell viability measurements. The MTT assay was first developedby Mosmann (See, e.g., Mosmann, J. Immunol. Meth., 65:55 (1983)). It isa simple colorimetric assay numerous laboratories have utilized forobtaining toxicity results (See e.g., Kuhlmann et al., Arch. Toxicol.,72:536 (1998)). Briefly, the mitochondria produce ATP to providesufficient energy for the cell. In order to do this, the mitochondriametabolize pyruvate to produce acetyl CoA. Within the mitochondria,acetyl CoA reacts with various enzymes in the tricarboxylic acid cycleresulting in subsequent production of ATP. One of the enzymesparticularly useful in the MTT assay is succinate dehydrogenase. MTT(3-(4,5-dimethylthiazol-2-yl)-2 diphenyl tetrazolium bromide) is ayellow substrate that is cleaved by succinate dehydrogenase forming apurple formazan product. The alteration in pigment identifies changes inmitochondria function. Nonviable cells are unable to produce formazan,and therefore, the amount produced directly correlates to the quantityof viable cells. Absorbance at 540 nm is utilized to measure the amountof formazan product.

The results of the in vitro tests can be compared to in vivo toxicitytests in order to extrapolate to live animal conditions. Typically,acute toxicity from a single dose of the substance is assessed. Animalsare monitored over 14 days for any signs of toxicity (increasedtemperature, breathing difficulty, death, etc). Traditionally, thestandard of acute toxicity is the median lethal dose (LD₅₀), which isthe predicted dose at which half of the treated population would bekilled. The determination of this dose occurs by exposing test animalsto a geometric series of doses under controlled conditions. Other testsinclude subacute toxicity testing, which measures the animal's responseto repeated doses of the nanodevice for no longer than 14 days.Subchronic toxicity testing involves testing of a repeated dose for 90days. Chronic toxicity testing is similar to subchronic testing but maylast for over a 90-day period. In vivo testing can also be conducted todetermine toxicity with respect to certain tissues. For example, in someembodiments of the present invention tumor toxicity (i.e., effect of thecompositions of the present invention on the survival of tumor tissue)is determined (e.g., by detecting changes in the size and/or growth oftumor tissues).

IX. Gene Therapy Vectors

In some embodiments of the present invention, the dendrimer conjugatescomprise transgenes for delivery and expression to a target cell ortissue, in vitro, ex vivo, or in vivo. In such embodiments, rather thancontaining the actual protein, the dendrimer complex comprises anexpression vector construct containing, for example, a heterologous DNAencoding a gene of interest and the various regulatory elements thatfacilitate the production of the particular protein of interest in thetarget cells.

In some embodiments, the gene is a therapeutic gene that is used, forexample, to treat cancer, to replace a defective gene, or a marker orreporter gene that is used for selection or monitoring purposes. In thecontext of a gene therapy vector, the gene may be a heterologous pieceof DNA. The heterologous DNA may be derived from more than one source(i.e., a multigene construct or a fusion protein). Further, theheterologous DNA may include a regulatory sequence derived from onesource and the gene derived from a different source.

Tissue-specific promoters may be used to effect transcription inspecific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate. Similarly, promoters may be used to target gene expression inother tissues (e.g., insulin, elastin amylase, pdr-1, pdx-1 andglucokinase promoters target to the pancreas; albumin PEPCK, HBVenhancer, alpha fetoproteinapolipoprotein C, alpha-1 antitrypsin,vitellogenin, NF-AB and transthyretin promoters target to the liver;myosin H chain, muscle creatine kinase, dystrophin, calpain p94,skeletal alpha-actin, fast troponin 1 promoters target to skeletalmuscle; keratin promoters target the skin; sm22 alpha; SM-.alpha.-actinpromoters target smooth muscle; CFTR; human cytokeratin 18 (K18);pulmonary surfactant proteins A, B and Q CC-10; P1 promoters target lungtissue; endothelin-1; E-selectin; von Willebrand factor; KDR/flk-1target the endothelium; tyrosinase targets melanocytes).

The nucleic acid may be either cDNA or genomic DNA. The nucleic acid canencode any suitable therapeutic protein. Preferably, the nucleic acidencodes a tumor suppressor, cytokine, receptor, inducer of apoptosis, ordifferentiating agent. The nucleic acid may be an antisense nucleicacid. In such embodiments, the antisense nucleic acid may beincorporated into the nanodevice of the present invention outside of thecontext of an expression vector.

In preferred embodiments, the nucleic acid encodes a tumor suppressor,cytokines, receptors, or inducers of apoptosis. Suitable tumorsuppressors include BRCA1, BRCA2, C-CAM, p16, p211 p53, p73, or Rb.Suitable cytokines include GMCSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,β-interferon, γ-interferon, or TNF. Suitable receptors include CFTR,EGFR, estrogen receptor, IL-2 receptor, or VEGFR. Suitable inducers ofapoptosis include AdE1B, Bad, Bak, Bax, Bid, Bik, Bim, Harakiri, orICE-CED3 protease.

X. Methods of Combined Therapy

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. Dendrimer conjugates of the present inventionprovide means of ameliorating this problem by effectively administeringa combined therapy approach. However, it should be noted thattraditional combination therapy may be employed in combination with thenanodevices of the present invention. For example, in some embodimentsof the present invention, dendrimer conjugates may be used before,after, or in combination with the traditional therapies.

To kill cells, inhibit cell growth, or metastasis, or angiogenesis, orotherwise reverse or reduce the malignant phenotype of tumor cells usingthe methods and compositions of the present invention in combinationtherapy, one contacts a “target” cell with the nanodevices compositionsdescribed herein and at least one other agent. These compositions areprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theimmunotherapeutic agent and the agent(s) or factor(s) at the same time.This may be achieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes, for example, an expressionconstruct and the other includes a therapeutic agent.

Alternatively, dendrimer conjugate treatment may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and immunotherapy are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and nanodevice would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that cells are contacted with both modalities within about12-24 hours of each other and, more preferably, within about 6-12 hoursof each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2 to 7)to several weeks (1 to 8) lapse between the respective administrations.

In some embodiments, more than one administration of theimmunotherapeutic composition of the present invention or the otheragent are utilized. Various combinations may be employed, where thedendrimer is “A” and the other agent is “B”, as exemplified below:A/B/A, B/A/B, B/B/A, A/A/B, B/A/A, A/B/B, B/B/B/A, B/B/A/B, A/A/B/B,A/B/A/B, A/B/B/A, B/B/A/A, B/A/B/A, B/A/A/B, B/B/B/A, A/A/A/B, B/A/A/A,A/B/A/A, A/A/B/A, A/B/B/B, B/A/B/B, B/B/A/B.

Other combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill or disable the cell.

Other factors that may be used in combination therapy with the dendrimerconjugates of the present invention include, but are not limited to,factors that cause DNA damage such as .gamma.-rays, X-rays, and/or thedirected delivery of radioisotopes to tumor cells. Other forms of DNAdamaging factors are also contemplated such as microwaves andUV-irradiation. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 weeks), to singledoses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes varywidely, and depend on the half-life of the isotope, the strength andtype of radiation emitted, and the uptake by the neoplastic cells. Theskilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

In preferred embodiments of the present invention, the regional deliveryof the dendrimer conjugates to patients with cancers is utilized tomaximize the therapeutic effectiveness of the delivered agent.Similarly, the chemo- or radiotherapy may be directed to particular,affected region of the subjects body. Alternatively, systemic deliveryof the immunotherapeutic composition and/or the agent may be appropriatein certain circumstances, for example, where extensive metastasis hasoccurred.

In addition to combining the dendrimer conjugates with chemo- andradiotherapies, it also is contemplated that traditional gene therapiesare used. For example, targeting of p53 or p16 mutations along withtreatment of the dendrimer conjugates provides an improved anti-cancertreatment. The present invention contemplates the co-treatment withother tumor-related genes including, but not limited to, p21, Rb, APC,DCC, NF-I, NF-2, BCRA2, p16, FHIT, WT-I, MEN-I, MEN-H, BRCA1, VHL, FCC,MCC, ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp, hst, bcl, andabl.

In vivo and ex vivo treatments are applied using the appropriate methodsworked out for the gene delivery of a particular construct for aparticular subject. For example, for viral vectors, one typicallydelivers 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10″ or1×10¹² infectious particles to the patient. Similar figures may beextrapolated for liposomal or other non-viral formulations by comparingrelative uptake efficiencies.

An attractive feature of the present invention is that the therapeuticcompositions may be delivered to local sites in a patient by a medicaldevice. Medical devices that are suitable for use in the presentinvention include known devices for the localized delivery oftherapeutic agents. Such devices include, but are not limited to,catheters such as injection catheters, balloon catheters, double ballooncatheters, microporous balloon catheters, channel balloon catheters,infusion catheters, perfusion catheters, etc., which are, for example,coated with the therapeutic agents or through which the agents areadministered; needle injection devices such as hypodermic needles andneedle injection catheters; needleless injection devices such as jetinjectors; coated stents, bifurcated stents, vascular grafts, stentgrafts, etc.; and coated vaso-occlusive devices such as wire coils.

Exemplary devices are described in U.S. Pat. Nos. 5,935,114; 5,908,413;5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719;5,851,228; 5,843,089; 5,800,519; 5,800,508; 5,800,391; 5,354,308;5,755,722; 5,733,303; 5,866,561; 5,857,998; 5,843,003; and 5,933,145;the entire contents of which are incorporated herein by reference.Exemplary stents that are commercially available and may be used in thepresent application include the RADIUS (SCIMED LIFE SYSTEMS, Inc.), theSYMPHONY (Boston Scientific Corporation), the Wallstent (SchneiderInc.), the PRECEDENT H (Boston Scientific Corporation) and the NIR(Medinol Inc.). Such devices are delivered to and/or implanted at targetlocations within the body by known techniques.

XI. Photodynamic Therapy

In some embodiments, the therapeutic complexes of the present inventioncomprise a photodynamic compound and a targeting agent that isadministered to a patient. In some embodiments, the targeting agent isthen allowed a period of time to bind the “target” cell (e.g. about 1minute to 24 hours) resulting in the formation of a target cell-targetagent complex. In some embodiments, the therapeutic complexes comprisingthe targeting agent and photodynamic compound are then illuminated(e.g., with a red laser, incandescent lamp, X-rays, or filteredsunlight). In some embodiments, the light is aimed at the jugular veinor some other superficial blood or lymphatic vessel. In someembodiments, the singlet oxygen and free radicals diffuse from thephotodynamic compound to the target cell (e.g. cancer cell or pathogen)causing its destruction.

XII. Pharmaceutical Formulations

Where clinical applications are contemplated, in some embodiments of thepresent invention, the dendrimer conjugates are prepared as part of apharmaceutical composition in a form appropriate for the intendedapplication. Generally, this entails preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals. However, in some embodiments of thepresent invention, a straight dendrimer formulation may be administeredusing one or more of the routes described herein.

In preferred embodiments, the dendrimer conjugates are used inconjunction with appropriate salts and buffers to render delivery of thecompositions in a stable manner to allow for uptake by target cells.Buffers also are employed when the dendrimer conjugates are introducedinto a patient. Aqueous compositions comprise an effective amount of thedendrimer conjugates to cells dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase “pharmaceutically or pharmacologically acceptable”refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. Except insofar as any conventional media or agent is incompatiblewith the vectors or cells of the present invention, its use intherapeutic compositions is contemplated. Supplementary activeingredients may also be incorporated into the compositions.

In some embodiments of the present invention, the active compositionsinclude classic pharmaceutical preparations. Administration of thesecompositions according to the present invention is via any common routeso long as the target tissue is available via that route. This includesoral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal or intravenous injection.

The active dendrimer conjugates may also be administered parenterally orintraperitoneally or intratumorally. Solutions of the active compoundsas free base or pharmacologically acceptable salts are prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

In some embodiments, a therapeutic agent is released from dendrimerconjugates within a target cell (e.g., within an endosome). This type ofintracellular release (e.g., endosomal disruption of alinker-therapeutic conjugate) is contemplated to provide additionalspecificity for the compositions and methods of the present invention.In some embodiments, the dendrimer conjugates of the present inventioncontain between 100-150 primary amines on the surface. Thus, the presentinvention provides dendrimers with multiple (e.g., 100-150) reactivesites for the conjugation of linkers and/or functional groupscomprising, but not limited to, therapeutic agents, targeting agents,imaging agents and biological monitoring agents.

The compositions and methods of the present invention are contemplatedto be equally effective whether or not the dendrimer conjugates of thepresent invention comprise a fluorescein (e.g. FITC) imaging agent.Thus, each functional group present in a dendrimer composition is ableto work independently of the other functional groups. Thus, the presentinvention provides dendrimer conjugates that can comprise multiplecombinations of targeting, therapeutic, imaging, and biologicalmonitoring functional groups.

The present invention also provides a very effective and specific methodof delivering molecules (e.g., therapeutic and imaging functionalgroups) to the interior of target cells (e.g., cancer cells). Thus, insome embodiments, the present invention provides methods of therapy thatcomprise or require delivery of molecules into a cell in order tofunction (e.g., delivery of genetic material such as siRNAs).

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The carrier may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial anantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it may be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, dendrimer conjugates are administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution is suitably buffered, if necessary,and the liquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intraperitonealadministration. For example, one dosage could be dissolved in 1 ml ofisotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). In some embodiments of the present invention, the activeparticles or agents are formulated within a therapeutic mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose orso. Multiple doses may be administered.

Additional formulations that are suitable for other modes ofadministration include vaginal suppositories and pessaries. A rectalpessary or suppository may also be used. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or the urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1%-2%. Vaginal suppositories or pessaries areusually globular or oviform and weighing about 5 g each. Vaginalmedications are available in a variety of physical forms, e.g., creams,gels or liquids, which depart from the classical concept ofsuppositories. In addition, suppositories may be used in connection withcolon cancer. The dendrimer conjugates also may be formulated asinhalants for the treatment of lung cancer and such like.

XIII. Method of Treatment or Prevention of Cancer and PathogenicDiseases

In some embodiments of the present invention methods and compositionsare provided for the treatment of tumors in cancer therapy. It iscontemplated that the present therapy can be employed in the treatmentof any cancer for which a specific signature has been identified orwhich can be targeted. Cell proliferative disorders, or cancers,contemplated to be treatable with the methods of the present inventioninclude human sarcomas and carcinomas, including, but not limited to,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,Ewing's tumor, lymphangioendotheliosarcoma, synovioma, mesothelioma,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilns' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease.

It is contemplated that the present therapy can be employed in thetreatment of any pathogenic disease for which a specific signature hasbeen identified or which can be targeted for a given pathogen. Examplesof pathogens contemplated to be treatable with the methods of thepresent invention include, but are not limited to, Legionellapeomophilia, Mycobacterium tuberculosis, Clostridium tetani, Hemophilusinfluenzae, Neisseria gonorrhoeae, Treponema pallidum, Bacillusanthracis, Vibrio cholerae, Borrelia burgdorferi, Cornebacteriumdiphtheria, Staphylococcus aureus, human papilloma virus, humanimmunodeficiency virus, rubella virus, polio virus, and the like.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1

Experiments were conducted during development of embodiments of theinvention in order to analyze and characterize various schemes forgenerating dendrimer conjugates wherein a dendrimer is conjugated to oneor more linkers that comprise multiple sites for binding (e.g., covalentbinding) moieties. A drug releasing mechanism for esterase sensitivelinker-dendrimer conjugates was analyzed (See e.g., FIG. 10). In someembodiments, once the ester bond is cleaved (e.g., by esterases (e.g.,present at a target site (e.g., intrinsic to the target))), irreversibledecomposition of the linkers leads to release of drug and/or therapeuticagent (e.g., at the target site).

Three elimination linkers (See FIG. 11, A-C) designed for esterasetriggered cleavage were synthesized. In some embodiments, the linkersare conjugated to a therapeutic agent and/or to a dendrimer (e.g., G5dendrimer).

Example 2 Synthesis of Esterase Sensitive Linker 11A

A synthesis scheme of a dendrimer (e.g., G5 PAMAM dendrimer) conjugatedto a therapeutic agent (e.g., TAXOL) with an esterase sensitive linker(esterase sensitive elimination linker 11A) is shown below.

Step 1:

A 50 mL, solution of Boc-β-alanine (500 mg, 2.64 mmol), EDC (506 mg,2.64 mmol), and DMAP (322 mg, 2.64 mmol) in methylene chloride wasstirred at 0° C. for 20 min. 4-Hydroxybenzaldehyde (323 mg, 2.64 mmol)was then added slowly. The reaction mixture was stirred at 0° C. for 2hour before it was warmed to RT and continued for over night. Thereaction mixture was then diluted with EtOAc and H₂O and extractive workup to give a crude product which was purified by silica gelchromatography to afford a clear oil (712 mg, 92%).

MS (EI) m/e=294 (M+1)

Step 2:

The aldehyde 1 (775 mg, 2.64 mmol) was dissolved in 40 mL of dry THF andwas cooled to 0° C. Boran in THF (1N solution, 2.64 mL) was addeddropwise. The reaction mixture was for 2 h. MeOH (5 mL) was added slowlyand the reaction mixture was warmed to RT in 1 h. Solvent was evaporatedand the product was purified by chromatograph to afford the product as awhite solid (625 mg, 80%).

MS (EI) m/e=296 (M+1)

Step 3:

The benzyl alcohol 2 (456 mg, 1.54 mmol) and p-nitrobenzyl chloroformate(934 mg, 4.63 mmol) were dissolved in 20 mL of methylene chloride.Pyridine (0.42 mL, 5.19 mmol) was added. White precipitate was formedduring the addition process. The reaction mixture was stirred at RT overnight. The reaction mixture was then diluted with EtOAc and water.Layers were separated and the aqueous layer was extracted with EtOAc×3.Combined organic solution was washed with 1N HCl, sat'd NaHCO₃ andbrine. The crude mixture was purified by silica gel chromatographyeluting with 15-25% EtOAc in Hexanes to afford the product as clear oil(520 mg, 73%).

MS (EI) m/e=461 (M+1)

Step 4:

Taxol (9.3 mg, 0.0103575 mmol) and 3 (4.75 mg, 0.0103575 mmol) weredissolved in dry methylene chloride (1 mL). A solution of DMAP (2.5 mg,0.02715 mmol) in methylene chloride (1 mL) was added dropwise at roomtemperature. After addition, a light yellow color appeared. The reactionmixture was allowed to stir at room temperature for 3 hours when TLCindicated the reaction was complete. The reaction mixture was extractedwith methylene chloride and water. The organic layers were combined anddried over MgSO₄. Solvent was evaporated after filtration. The residuewas purified by column chromatography (silica gel, EtOAc:Hexanes 1:1)and pure product (10 mg, yield 82%) was obtained.

MS (EI) m/e=1197.5 (M+Na).

Step 5:

Taxol-linker conjugate 4 (10.0 mg, 0.008514 mmol) was dissolved inmethylene chloride (1 mL). To above solution was added TFA (120 μL). Thereaction mixture was stirred at room temperature and was checked withTLC until the reaction was complete in 20 minutes. The solvent wasevaporated and the residue was purified by column chromatography (silicagel, CH₂Cl₂:MeOH 10:1). Product 5 was isolated as a white solid (8.0 mg,yield 87.4%).

MS (EI) m/e=1075.4 (M+H).

Step 6:

G5-Ac-FI-FA-COOH (6), prepared as reported previously, (15.3 mg,0.0004636 mmol) was dissolved in H₂O (5.2 mL), EDM (10.6 mg, 0.03488mmol) was added. The reaction mixture was stirred for 2 hour at roomtemperature. A solution of 5 (10 mg, 0.0093 mmol) in DMF (4.3 mL) andDMSO (3.4 mL) was added dropwise. The reaction was allowed to stir atroom temperature for three days. The solvent was removed by membranefiltration through a 10,000 MWCO membrane. The residue was furtherpurified by passing through a Sephdex G-25 column and extensively washedwith PBS buffer and water. Lyophilization gave final product 7 as orangecolored solid (15.1 mg, yield 90%).

Example 3 Synthesis of Esterase Sensitive Linker 11B

A synthesis scheme of a dendrimer (e.g., G5 PAMAM dendrimer) conjugatedto a therapeutic agent (e.g., Taxol) with an esterase sensitive linker(esterase sensitive elimination linker 11B) is shown below.

Step 1:

A mixture of 5-formyl-2-hydroxybenzoic acid 8 (1.66 g, 10 mmol),mono-Boc-ethylene diamine (1.60 g, 10 mmol), EDC methiodide (2.97 g, 10mmol), and HOBT (1.35 g, 10 mmol) was dissolved in 40 mL of DMF at 0° C.The solution was stirred at this temperature for 1 h before it waswarmed to RT. Stirring was continued for over night. The orange-yellowcolored reaction mixture was cooled to 0° C. Triethylamine (2.8 mL, 20mmol) was added followed by pivaloyl chloride (2.5 mL, 20 mmol). Thereaction mixture was stirred for 2 hours and it was quenched by additionof 50 ml of water. EtOAc (200 mL) was added and the layers wereseparated. The aqueous layer was extracted with EtOAc×3. The combinedorganics was washed with 1N HCl, saturated NaHCO₃ solution, and brinesequentially and was dried with MgSO₄. After solvent was evaporated, theresidue was purified by silica gel chromatography to afford the product9 as a pale yellow solid (3.33 g, 85% 2 steps).

MS (EI) ink=xxx (M+1)

Step 2:

The aldehyde 9 (2.04 g, 5.20 mmol) was dissolved in 60 mL of dry THF andwas cooled to 0° C. Boran in THF (1N solution, 5.46 mL) was addeddropwise. The reaction mixture was for 2 h. MeOH (10 mL) was addedslowly and the reaction mixture was warmed to RT in 2 h. Solvent wasevaporated and the product was purified by chromatograph to afford theproduct as a white solid (2.02 g, 98%).

MS (EI) m/e=xxx (M+1)

Step 3:

The benzyl alcohol 10 (1.185 g, 3.0 mmol) and p-nitrobenzylchloroformate (908 mg, 4.50 mmol) were dissolved in 30 mL of methylenechloride. Pyridine (0.49 mL, 6.0 mmol) was added. White precipitate wasformed during the addition process. The reaction mixture was stirred atRT over night. The reaction mixture was then diluted with EtOAc andwater. Layers were separated and the aqueous layer was extracted withEtOAc×3. Combined organic solution was washed with 1N HCl, sat'd NaHCO₃and brine. The crude mixture was purified by silica gel chromatographyeluting with 15-25% EtOAc in Hexanes to afford the product 11 as whitesolid (1.38 g, 82%).

MS (EI) m/e=560 (M+1)

Step 4:

In a 5 mL round bottle flask, taxol (20 mg, 0.02227 mmol) and linker 11(35.8 mg, 0.02810 mmol) were dissolved in dry methylene chloride (2 mL).A solution of DMAP (5.7 mg, 0.04666 mmol) in methylene chloride (1 mL)was added dropwise at room temperature. After addition, a light yellowappeared. The reaction mixture was allowed to stir at room temperaturefor 3 hours. The reaction was monitored with TLC until the reaction wascomplete. The reaction mixture was extracted with methylene chloride andwater. The organic layer was collected and dried over MgSO₄ andevaporated. The residue was purified by column chromatography (silicagel, AcOEt:Hexanes 1:1) and pure product (25.3 mg, yield 89%) wasobtained.

MS (EI) m/e=1296.5 (M+Na).

Step 5:

To the taxol-linker conjugate 12 (12 mg, 0.009426 mmol) in methylenechloride (1 mL) was added TFA (120 μL). The reaction mixture was stirredat room temperature for 20 minutes and was checked with TLC until thereaction was complete. The solvent was evaporated and the residue waspurified by column chromatography (silica gel, CH₂Cl₂:MeOH 10:1).Product 13 was isolated as a white solid (9.8 mg, yield 88%).

MS (EI) m/e=1174.5 (M+H).

Step 6:

G5-Ac-FI-FA-COOH (6), prepared as reported previously, (13.8 mg,0.00041818 mmol) was dissolved in H₂O (5.2 mL), EDM (9.32 mg, 0.03136mmol) was added. The reaction mixture was stirred for 2 hour at roomtemperature. A solution of 13 (9.8 mg, 0.0093 mmol) in DMF (4.3 mL) andDMSO (3.4 mL) was added dropwise. The reaction was allowed to stir atroom temperature for three days. The solvent was removed by membranefiltration through a 10,000 MWCO membrane. The residue was furtherpurified by passing through a Sephdex G-25 column and extensively washedwith PBS buffer and water. Lyophilization gave final product 14 asorange colored solid (15.1 mg, yield 87%).

Example 4 Synthesis of Esterase Sensitive Linker 11C

A synthesis scheme of a dendrimer (e.g., G5 PAMAM dendrimer) conjugatedto a therapeutic agent (e.g., Taxol) with an esterase sensitive linker(esterase sensitive elimination linker 11C) is shown below.

Example 5 Additional Self-Immorlative Linkers

The present invention is not limited by the type of self-immorlativelinkers utilized. For example, cyclization based linkers can be used.Although a mechanism is not necessary to practice the present inventionand the present invention is not limited to any particular mechanism, insome embodiments, a mechanism as shown below is utilized in a conjugateof the present invention:

Thus, in some embodiments, the present invention provides synthesis ofdendrimer conjugates utilizing cyclization linkers (e.g., designed asesterase cleavage substrates) as shown below:

In some embodiments, the present invention provides syntheses of linkersC and D as shown below.

Example 6 Characterization of Dendrimer Conjugates

Experiments were conducted during development of embodiments of theinvention in order to characterize release of drug from a dendrimerconjugate comprising a linker-drug component. The linker-drug componentswere characterized under esterase incubation conditions, utilizing HPLCas an analytical tool to monitor drug release. This approach provides anassessment regarding structural influences of the linkers. For example,characteristics of drug release from a linker (e.g., in the absence of adendrimer) provides information regarding drug release from a linkerconjugated to a dendrimer.

For example, the experiments were conducted to characterize thefollowing two conjugates:

First Generation Linker-Drug(-Dendrimer) Conjugates

When incubated with pig liver esterase for 2 hours, conjugate B showedminimal release and conjugate A showed ˜11% release. Furthermore,conjugate A showed around 40% release at 24 h (See, e.g., FIG. 12).

Example 7 Second Generation Linkers

Characterization of linkers as described in Example 6 indicated thatsteric hindrance issues were inhibiting release of a therapeutic fromthe conjugates (e.g., due, in some embodiments, to inaccessibility ofesterase to the linker). Based on this data, alternative approaches weregenerated and characterized. For example, in some embodiments, in orderto relieve steric hindrance, lengths of the linkers were extended. Thus,the present invention provides additional, “second generation” linkersas described below.

Second Generation Linker-Drug(-Dendrimer) Conjugates Rational

Thus, in some embodiments, the present invention provides conjugates andmethods of synthesizing and utilizing (e.g., therapeutically) the samewith extended linkages as shown in FIG. 13. In some embodiments thepresent invention provides conjugates as shown in FIGS. 14-16.

Example 8 Hypoxia Induced Linkers

The present invention also provides dendrimers comprising small moleculelinkers triggered by hypoxic environments (e.g., in and/or around cancercells). In some embodiments, a dendrimer of the present inventioncomprises a indolequinone linker. In some embodiments, a dendrimercomprising a hypoxia cleavable linker is generated according to thesynthesis scheme shown in FIG. 29. In some embodiments, a dendrimercomprising a hypoxia cleavable linker is generated according to thesynthesis scheme shown in FIG. 30. The present invention is not limitedto any particular mechanism of release of a therapeutic agent from adendrimer comprising a linker triggered by a hypoxic environment.Indeed, a variety of mechanisms are contemplated including, but notlimited to, a mechanism shown in FIG. 31.

In particular, experiments conducted during the course of development ofembodiments for the present invention demonstrated that incubation of a[dendrimer—indolequinone linker—Naloxone prodrug] with either freshfrozen plasma or a reductive results in release of the Naloxone prodrugunder hypoxic conditions, but not under normoxic conditions. Indeed,FIG. 32 shows release of Naloxone from [dendrimer-indolequinonelinker-Naloxone prodrug] using the reductive enzyme DT-diaphorase. FIG.33 shows release of Naloxone from [dendrimer-indolequinonelinker-Naloxone prodrug] in human plasma under hypoxic conditions, butnot under normoxic conditions. FIG. 34 shows hypoxia-induced releasekinetics for Naloxone from [dendrimer-indolequinone linker-Naloxoneprodrug] met or exceeded 6 mg Naloxone/hour at pO2 of 18 mmHg withinfresh frozen plasma.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the presentinvention.

We claim:
 1. A dendrimer conjugate comprising a linker, wherein saidlinker is selected from the group consisting of an elimination linker, acyclization linker, a glucoronidase sensitive linker, a branchedself-elimination linker, a heteroaromatic nitrogen containing compoundlinker, and a hypoxia induced linker, wherein said linker is conjugatedto a G5 PAMAM dendrimer and at least one ligand selected from the groupconsisting of a therapeutic agent, an imaging agent, and a targetingagent.
 2. The dendrimer conjugate of claim 1, wherein said G5 PAMAMdendrimer is conjugated to at least one ligand selected from the groupconsisting of a therapeutic agent, an imaging agent, and a targetingagent.
 3. The dendrimer conjugate of claim 1, wherein said eliminationlinker is a 1,4 elimination linker.
 4. The dendrimer conjugate of claim1, wherein said elimination linker is a 1,6 elimination linker.
 5. Thedendrimer conjugate of claim 1, wherein said hypoxia induced linker isesterase sensitive.
 6. The dendrimer of claim 1, wherein said hypoxiainduced linker is indolequinone.
 7. The dendrimer conjugate of claim 1,wherein said therapeutic agent is Naloxone.
 8. The dendrimer of claim 1,wherein said imaging agent is selected from the group consisting of aradioactive label, fluorescein isothiocyanate (FITC), 6-TAMARA, acridineorange, and cis-parinaric acid.
 9. The dendrimer of claim 1, whereinsaid targeting agent is selected from the group consisting of an agentbinding a receptor selected from the group consisting of CFTR, EGFR,estrogen receptor, FGR2, folate receptor, IL-2 receptor, and VEGFR; anantibody that binds to a polypeptide selected from the group consistingof p53, Mucl, a mutated version of p53 that is present in breast cancer,HER-2, T and Tn haptens in glycoproteins of human breast carcinoma, andMSA breast carcinoma glycoprotein; an antibody selected from the groupconsisting of human carcinoma antigen, TP1 and TP3 antigens fromosteocarcinoma cells, Thomsen-Friedenreich (TF) antigen fromadenocarcinoma cells, KC-4 antigen from human prostrate adenocarcinoma,human colorectal cancer antigen, CA125 antigen from cystadenocarcinoma,DF3 antigen from human breast carcinoma, and p97 antigen of humanmelanoma, carcinoma or orosomucoid-related antigen; transferrin; and asynthetic tetanus toxin fragment.
 10. The dendrimer of claim 1, whereinsaid therapeutic agent is selected from the group consisting of achemotherapeutic agent, an anti-oncogenic agent, an anti-angiogenicagent, a tumor suppressor agent, an anti-microbial agent, an expressionconstruct comprising a nucleic acid encoding a therapeutic protein, apain relief agent, a pain relief agent antagonist, an agent designed totreat arthritis, an agent designed to treat inflammatory bowel disease,an agent designed to treat an autoimmune disease, and an agent designedto treat inflammatory pelvic disease.